Modulators of ATP-binding cassette transporters

ABSTRACT

The present invention relates to modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including CF Transmembrane Regulator (“CFTR”), compositions thereof, and methods therewith. The present invention also relates to methods of treating ABC transporter mediated diseases using such modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/174,844, filed Jul. 1, 2011, which is a divisional of U.S. patentapplication Ser. No. 10/936,448, now U.S. Pat. No. 7,973,169, filed Sep.7, 2004, which claims priority to U.S. Provisional patent applicationNo. 60/500,444, filed Sep. 6, 2003, the entire contents of allapplications being incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including CF TransmembraneRegulator (“CFTR”), compositions thereof, and methods therewith. Thepresent invention also relates to methods of treating ABC transportermediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a group of membrane transporter proteins that playa major role in the transport and protection of cells against a widevariety of pharmacological agents, potentially toxic drugs, andxenobiotics. ABC transporters are homologous membrane proteins that bindand use cellular adenosine triphosphate (ATP) for their specificactivities. Some of these transporters were discovered as multidrugresistance proteins (like the MDR1-P glycoprotein, or the multidrugresistance protein, MRP1), defending malignant cancer cells againstchemotherapeutic agents. Up until the present time, 48 Human ABCTransporters have been identified, and these have been arranged into 7families based on their sequence identity and function.

ABC transporters play a variety of important physiological roles withinthe body, as well as providing a defense against harmful compounds fromthe environment. Moreover they represent important potential drugtargets both in their own right, as well as, because in many casestherapeutic drugs are also transported out of the target cell by thesemolecules.

One of the members of the ABC transporter family, namely, CFTR, isbelieved be the chloride channel responsible for cAMP-mediated chloridesecretion in epithelial cells, and to play a key role in the secretionof chloride and maintenance of normal electrolyte transport throughoutthe body. CFTR is a protein of approximately 1480 amino acids made up oftwo repeated elements, each comprising six transmembrane segments and anucleotide-binding domain. The two repeats are separated by a large,polar, regulatory (R)-domain containing multiple potentialphosphorylation sites.

The gene associated with CFTR has been identified and sequenced (SeeGregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al.(1990) Nature 347:358-362), (Riordan, J. R. et al. (1989) Science245:1066-1073). A defect in this gene leads to cystic fibrosis(hereinafter “CF”), the most common fatal genetic disease in humansaffecting approximately one in every 2,500 infants born in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the chronic effects of CF, including chronic lungdestruction and death.

In patients with Cystic fibrosis, expression of the CF associated genein airway cells, leads to reduced cellular apical chloride conductancecausing an imbalance in ion and fluid transport. It is widely believedthat this leads to the abnormal mucus secretion in pancreatic ductulesand in the airways that ultimately results in the pulmonary infectionsand epithelial cell damage typically associated with disease progressionin CF. In addition to respiratory problems, CF patients typically sufferfrom gastrointestinal problems, and pancreatic insufficiency. Males arealmost uniformly infertile and fertility is decreased in females. Incontrast to the severe effects of two copies of the CF associated gene,individuals with a single copy of the CF associated gene exhibitincreased resistance to cholera and to dehydration resulting fromdiarrhea—perhaps explaining the relatively high frequency of the CF genewithin the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). At present, more than 1000 mutationsin the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/), but population studies haveindicated that the most common CF mutation, a deletion of the 3nucleotides that encode phenylalanine at position 508 of the CFTR aminoacid sequence, is associated with approximately 70% of the cases ofcystic fibrosis. The mutated CFTR protein is referred to as ΔF508.

It is believed that the deletion of residue 508 in ΔF508-CFTR preventsthe nascent protein from folding correctly, resulting in the inabilityof this mutant protein to exit the endoplasmic reticulum (hereinafter“ER”), and traffic to the plasma membrane. As a result, insufficientamounts of the mature protein are present at the plasma membrane andchloride transport within epithelial tissues is significantly reduced(Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Hence, the cellularphenomenon of defective ER processing of other protein/s like CFTR, bythe ER machinery, has been shown to be the underlying basis for a widerange of isolated and inherited diseases. The two ways that the ERmachinery can malfunction is either by loss of coupling to ER export ofthe proteins leading to degradation, or by the ER accumulation of thesedefective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp745-751 (1999); Shastry, B. S., et al., Neurochem. International, 43, pp1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp 211-222(2002); Morello, J P et al., TIPS, 21, pp. 466-469 (2000); Bross P., etal., Human Mut., 14, pp. 186-198 (1999)]. Studies have shown, however,that ΔF508-CFTR, when presented at the plasma membrane is functional asa cAMP-responsive Cl⁻ channel (Dalemans et al. (1991), Nature Lond. 354:526-528; Denning et al., supra.; Pasyk and Foskett (1995), J. Cell.Biochem. 270: 12347-50).

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl− channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to CF, modulation of CFTR activity may be beneficial forother diseases not directly caused by mutations in CFTR, such assecretory diseases and other protein folding diseases mediated by CFTR.These include, but are not limited to, chronic obstructive pulmonarydisease (hereinafter “COPD”), dry eye disease, and Sjögren's Syndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such asCystic Fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery, has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)].

The diseases associated with the first class of ER malfunction are CF(due to misfolded ΔF508-CFTR), hereditary emphysema (due toα1-antitrypsin; non Piz variants), hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are glycanosis CDGtype 1, Hereditary emphysema (due to α1-Antitrypsin (PiZ variant),Congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-Antichymotrypsin), Diabetes insipidus (DI),neurophyseal DI (due to Vasopressin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to Peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as Spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to Prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A) andStraussler-Scheinker syndrome (due to Prp processing defect).

In CF, chloride transport mediated by the CFTR is reduced resulting inthe abnormal mucus secretion that characterizes the disease. By contrastin secretory diarrheas epithelial water transport is dramaticallyincreased as a result of secretagogue activated chloride transport. Themechanism involves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, death and impairedgrowth.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). Sixteen million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrheal causing bacteria is enterotoxogenic E. coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

There is a need for correctors that enhance the density of CFTR in theplasma membrane by facilitating the migration of the CFTR thereto.

SUMMARY OF THE INVENTION

The present invention provides a method of modulating ABC transporteractivity, comprising the step of contacting said ABC transporter with acompound of formula I or formula I′:

or a pharmaceutically acceptable salt thereof;wherein:

Y′ is O, S, or NR;

p is 0-2;

X is a bond, O, S, S(O), S(O)₂, CF₂, CH₂, —CHOR—, —C(O)—, —O—C(O)—,—C(O)—O, —C(O)—NR, —NR—C(O)—, —NR—C(O)—O—, —O—C(O)—NR—, —NR—C(O)—NR—, orNR;

R is H, R², or R⁶;

A is aliphatic, aryl, heteroaryl, heterocyclic, or cycloalkyl;

C is a phenyl or 5-8 membered cycloaliphatic ring;

Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

wherein each A, B, and C is independently and optionally substitutedwith up to 4 substituents independently selected from R¹, R², R³, R⁴, orR⁵;

R^(L) is —OR^(A), —SR^(A), or —N(R^(AB))₂;

each R^(A) is independently hydrogen, C1-C6 aliphatic, or a 3-7 memberedcarbocyclic or heterocyclic ring, saturated or unsaturated ring, havingup to 3 heteroatoms selected from O, N, or S, wherein each R^(A) isoptionally substituted with up to 3 substituents independently selectedfrom R¹, R⁴ or R⁷,

each R^(AB) is independently hydrogen or C1-C6 aliphatic optionallysubstituted with up to 3 substituents independently selected from R¹, R⁴or R⁷;

wherein up to two methylene units in R^(A) or R^(AB) are optionallyreplaced with —CO—, —CS—, —COCO—, —CONR—, —CO₂—, —OCO—, —NRCO₂—, —O—,—NRCONR—, —OCONR—, —NRCO—, —S—, —SO, —SO₂—, —NR—, —SO₂NR—, NRSO₂—, or—NRSO₂NR; or

two R^(AB), taken together with the nitrogen atom, is a 3-7 memberedheterocyclic or heteroaryl ring containing up to 4 heteroatoms selectedfrom O, N, or S, wherein said ring is optionally substituted with up to2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

R^(M) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

each of X₁ and X₂ is independently selected from O, S, or NR;

R^(N) is C1-C6 aliphatic or phenyl, wherein R^(N) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R^(P) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

R^(Q) is C1-C6 aliphatic or aryl, wherein R^(Q) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(O)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶,C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵,C(NOR⁵)R⁶, C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶,NR⁶C(O)R⁵, NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶) R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic), or O-aliphatic;and

R⁸ is an amino-capping group.

The present invention also provides compositions comprising compounds offormula (I) and formula (I′), and methods of treating ABC transportermediated diseases using compounds of formula (I) and formula (I′).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneregulator or a mutation thereof capable of its regulator activity, inpart or full, including, but not limited to, ΔF508 CFTR and G551D CFTR(see, e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.”

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain or branched, hydrocarbon chain that is completelysaturated (alkyl) or is unsaturated (alkenyl or alkynyl). Unlessotherwise specified, an aliphatic group has 1 to 12 carbon atoms,preferably, 1-6 carbon atoms, and more preferably, 1-4 carbon atoms. Upto three, and preferably two, —CH₂— in said aliphatic may be replacedwith O, S, or —NR.

The term “alkylidene” as used herein means a straight-chain or branchedhydrocarbon chain that is completely saturated or unsaturated, and isconnected to the rest of the molecule through covalent bonds. Exemplaryalkylidene groups include methylene, ethylene, or propylene. Unlessotherwise specified, an alkylidene group has 1 to 12 carbon atoms,preferably, 1-6 carbon atoms, and more preferably, 1-4 carbon atoms.

The term “cycloaliphatic” means a saturated or partially unsaturatedmonocyclic or bicyclic hydrocarbon ring that has up to two points ofattachment to the rest of the molecule. Unless otherwise specified,preferred cycloaliphatic rings are 3-8 membered monocyclic rings, morepreferably 3-6, and even more preferably, 3, 5, or 6. Also preferred,unless otherwise specified, are 8-12 membered bicyclic hydrocarbonrings, more preferably 10 membered bicyclic hydrocarbon rings.

The term “heteroatom,” unless otherwise specified, means nitrogen,oxygen, or sulfur and includes any oxidized form of nitrogen and sulfur,and the quaternized form of any basic nitrogen. Also the term “nitrogen”includes a substitutable nitrogen of a heterocyclic ring. As an example,in a saturated or partially unsaturated ring having 0-3 heteroatomsselected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or as in N-substitutedpyrrolidinyl.

The term “unsaturated”, as used herein, means a double bond or a triplebond. Each such bond constitutes one unit of unsaturation.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclicand tricyclic aromatic carbocyclic ring systems. Unless otherwisespecified, preferred aryl rings have a total of five to fourteen ringmembers, wherein at least one ring, if bicyclic or tricyclic, in thesystem is aromatic and wherein each ring in the system contains up to 6ring members. The term “aryl” may be used interchangeably with the term“aryl ring”. Phenyl is an example of aryl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinmeans non-aromatic, monocyclic, bicyclic or tricyclic ring systemswherein one or more ring members is a heteroatom. Unless otherwisespecified, each ring in the system preferably contains 3 to 7 ringmembers with preferably 1-3 heteroatoms.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclicand tricyclic ring systems, wherein at least one ring in the system isaromatic, and at least one ring in the system contains one or moreheteroatoms. Unless otherwise specified, such ring systems preferablyhave a total of 5 to 15 ring members, wherein each ring in the systempreferably contains 3 to 7 ring members, with preferably 1-3heteroatoms. The term “heteroaryl” may be used interchangeably with theterm “heteroaryl ring” or the term “heteroaromatic”.

A combination of substituents or variables is permissible only if such acombination results in a stable or chemically feasible compound. Astable compound or chemically feasible compound is one that is notsubstantially altered when kept at a temperature of 40° C. or less, inthe absence of moisture or other chemically reactive conditions, for atleast a week.

The present invention provides a method of modulating ABC transporteractivity, comprising the step of contacting said ABC transporter with acompound of formula I or formula I′:

or a pharmaceutically acceptable salt thereof;wherein:

Y′ is O, S, or NR;

p is 0-2;

X is a bond, O, S, S(O), S(O)₂, CF₂, CH₂, —CHOR—, —C(O)—, —O—C(O)—,—C(O)—O, —C(O)—NR, —NR—C(O)—, —NR—C(O)—O—, —O—C(O)—NR—, —NR—C(O)—NR—, orNR;

R is H, R², or R⁶;

A is aliphatic, aryl, heteroaryl, heterocyclic, or cycloalkyl;

C is a phenyl or 5-8 membered cycloaliphatic ring;

Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

wherein each A, B, and C is independently and optionally substitutedwith up to 4 substituents independently selected from R¹, R², R³, R⁴, orR⁵;

R^(L) is —OR^(A), —SR^(A), or —N(R^(AB))₂;

each R^(A) is independently hydrogen, C1-C6 aliphatic, or a 3-7 memberedcarbocyclic or heterocyclic ring, saturated or unsaturated ring, havingup to 3 heteroatoms selected from O, N, or S, wherein each R^(A) isoptionally substituted with up to 3 substituents independently selectedfrom R¹, R⁴ or R⁷,

each R^(AB) is independently hydrogen or C1-C6 aliphatic optionallysubstituted with up to 3 substituents independently selected from R¹, R⁴or R⁷;

wherein up to two methylene units in R^(A) or R^(AB) are optionallyreplaced with —CO—, —CS—, —COCO—, —CONR—, —CO₂—, —OCO—, —NRCO₂—, —O—,—NRCONR—, —OCONR—, —NRCO—, —S—, —SO, —SO₂—, —NR—, —SO₂NR—, NRSO₂—, or—NRSO₂NR; or

two R^(AB), taken together with the nitrogen atom, is a 3-7 memberedheterocyclic or heteroaryl ring containing up to 4 heteroatoms selectedfrom O, N, or S, wherein said ring is optionally substituted with up to2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

R^(M) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

each of X₁ and X₂ is independently selected from O, S, or NR;

R^(N) is C1-C6 aliphatic or phenyl, wherein R^(N) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R^(P) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

R^(Q) is C1-C6 aliphatic or aryl, wherein R^(Q) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(R⁸)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶, C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵)R⁶,C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵,NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶) R⁶, N(O)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic, or O-aliphatic;and

R⁸ is an amino-capping group.

The term “amino capping group” refers to a suitable chemical group thatmay be attached to a nitrogen atom. The term “capping” refers to whenthe designated amino group is attached to a suitable chemical group(e.g., protecting group). Examples of suitable amino capping groups aredescribed in T. W. Greene et al., Protective Groups in OrganicSynthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); L. Paquette, ed. Encyclopedia of Reagents for Organic Synthesis,John Wiley and Sons (1995) and are exemplified in certain of thespecific compounds used in this invention.

According to a preferred embodiment, Y′ is S.

According to one embodiment, Y′ is O.

According to another embodiment, Y′ is NR. In one embodiment, R is H.Or, R is R². Or, R is R⁶.

According to another embodiment, p is 1. Or, p is 1 and X is attached tothe carbon adjacent to Y′ atom. Or, p is 1 and X is attached to thecarbon adjacent to the ring nitrogen atom.

According to another embodiment, p is 2.

According to another embodiment, X is a bond, O, S, CH₂, CF₂, CHOR,C(O)NR, C(O)O—, NRC(O), or NR. In certain embodiments, X is a bond, O,or CH₂. In other embodiments, X is CH₂.

According to another embodiment, A is an optionally substituted C3-C7cycloaliphatic ring. Preferably, A is an optionally substitutedcyclopropyl, cyclopentyl, or cyclohexyl.

According to another embodiment, A is optionally substituted(C1-C10)aliphatic. In certain embodiments, A is optionally substitutedmethyl, ethyl, propyl, butyl, pentyl, or hexyl.

According to another embodiment, A is optionally substituted C6-C10 arylring. In one embodiment, A is optionally substituted phenyl or naphthyl.

According to another embodiment, A is optionally substituted C5-C12heteroaryl ring. In certain embodiments, A is selected from optionallysubstituted triazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridinyl,thiadiazolyl, triazolyl, oxadiazolyl, isothiazolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, pyrrolyl, thienyl, furanyl,indolizinyl, indolyl, isoindolyl, benzofuranyl, benzo[b]thienyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, acridinyl, phenazinyl, phenothiazinyl,or phenoxazinyl.

According to another embodiment, A is optionally substituted C3-C12heterocyclic ring. In certain embodiments, A is selected from optionallysubstituted aziridine, oxirane, thiirane, pyrrolidyl, tetrahydrofuranyl,tetrahydrothienyl, dioxolanyl, pyrrolinyl, pyranyl, pyrazolinyl,pyrazolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,thiomorpholinyl, piperazinyl, 3H-indolyl, or indolinyl.

In some embodiments, A, X, and the ring attached thereto, takentogether, is selected from:

wherein:

R^(Ph) is independently R¹, R², or R³; and

r is 0-3.

X₅ is CH₂, C(O), or CHOR;

X₆ is O or NR²; and

R^(Ak) is C1-C6 aliphatic, optionally substituted with R¹, R², or R³.

In another embodiment, A, X, and the ring attached thereto, takentogether, is selected from any of the rings i to xviii, wherein thesulfur atom in each of the thiazole ring is replaced with an oxygen atom(to provide the corresponding oxazole).

According to another embodiment, each B is independently selected fromoptionally substituted C6-C10 aryl. In certain embodiments, each B is anoptionally substituted phenyl or naphthyl. Or, each B is anunsubstituted phenyl.

According to another embodiment, each B is independently selected fromoptionally substituted C5-C12 heteroaryl. In certain embodiments, each Bis independently an optionally substituted C5-C7 heteroaryl.

According to another embodiment, each B is independently selected fromtriazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridinyl, thiadiazolyl,triazolyl, oxadiazolyl, isothiazolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, pyrrolyl, thienyl, furanyl, indolizinyl, indolyl, isoindolyl,benzofuranyl, benzo[b]thienyl, 1H-indazolyl, benzimidazolyl,benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl,phthazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,indenyl, naphthyl, azulinyl, or anthracenyl.

According to another embodiment, R¹ is 1,2-methylene dioxy, or1,2-ethylenedioxy.

According to another embodiment, R¹ is R⁶, wherein R⁶ is straight chainor branched (C1-C6)alkyl or (C2-C6) alkenyl or alkynyl, optionallysubstituted with R⁷.

According to another embodiment, R¹ is (C1-C4 aliphatic)_(n)-Y, whereinn is 0 or 1, and Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶,NH₂, NHR⁶, N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ or OR⁶;

According to another embodiment, R¹ is selected from halo, CF₃, NH₂,NH(C1-C4 alkyl), NHC(O)CH₃, OH, O(C1-C4 alkyl), OPh, O-benzyl, S—(C1-C4alkyl), C1-C4 aliphatic, CN, methylenedioxy, ethylenedixoy, SO₂NH(C1-C4alkyl), or SO₂N(C1-C4 alkyl)₂.

According to another embodiment, R¹ is selected from methyl, n-propyl,i-propyl, t-butyl, halo, CF₃, NH₂, NH(CH₃), NHC(O)CH₃, OH, OCH₃, OPh,O-benzyl, S—(C₂H₅), S—CH₃, NO₂, CN, methylenedioxy, SO₂NH(n-propyl), orSO₂N (n-propyl)₂.

According to one embodiment, R² is a straight chain or branched(C1-C6)alkyl or (C2-C6) alkenyl or alkynyl, optionally substituted withR¹, R⁴, or R⁵. In certain embodiments, R² is a straight chain orbranched (C1-C4)alkyl or (C2-C4) alkenyl or alkynyl, optionallysubstituted with R¹, R⁴, or R⁵.

According to another embodiment, R³ is optionally substituted phenyl,napthyl, C5-C10 heteroaryl or C3-C7 heterocyclyl. In certainembodiments, R³ is an optionally substituted phenyl, C5-C6 heteroaryl,or C3-C6 heterocyclyl.

According to one embodiment, R⁴ is selected from OR⁵, OR⁶, SR⁵, SR⁶,NR⁵COR⁵, NR⁵COR⁶, NR⁶COR⁵, or NR⁶COR⁶.

According to one embodiment, R⁵ is C5-C6 cycloalkyl, C6 or C10 aryl,C5-C10 heteroaryl or C3-C7 heterocyclyl, optionally substituted with upto 2 R¹. In certain embodiments, R⁵ is an optionally substitutedcyclohexyl, phenyl, C5-C6 heteroaryl, or C3-C6 heterocyclyl.

According to one embodiment, R⁶ is H.

According to another embodiment, R⁶ is a straight chain or branched(C1-C6)alkyl or (C2-C6 alkenyl) or alkynyl, optionally substituted withR⁷.

According to another embodiment, R⁶ is a straight chain or branched(C1-C6)alkyl or (C2-C6 alkenyl) or alkynyl.

According to one embodiment, R⁷ is C5-C6 cycloalkyl, phenyl, naphthyl,C5-C10 heteroaryl or C3-C7 heterocyclyl, optionally substituted withstraight chain or branched (C1-C6)alkyl or (C2-C6 alkenyl) or alkynyl.Or, R⁷ is C5-C6 cycloalkyl, phenyl, naphthyl, C5-C10 heteroaryl or C3-C7heterocyclyl, optionally substituted with 1-2-methylenedioxy,1,2-ethylenedioxy, or (CH₂)_(n)—Z. In certain embodiments, R⁷ is anoptionally substituted cyclohexyl, phenyl, C5-C6 heteroaryl, or C3-C6heterocyclyl.

According to a preferred embodiment, R⁸ is acetyl, arylsulfonyl oralkylsulfonyl.

In another embodiment, Q in compounds of formula I is selected from:

In one embodiment, the present invention provides compounds havingformula I-a:

In certain embodiments, p is 1. Or, p is 2.

In certain embodiments, X is a bond, O, CH₂, CHOH, C(O), or C(O)O. Or, Xis a bond, O, or CH₂.

In certain embodiments, p is 1 and X is a bond. In one embodiment, p is1, and X-A is attached to the carbon adjacent to the ring nitrogen atom.Or, p is 1, and X-A is attached to the carbon adjacent to the Y′.

In certain embodiments, p is 1, X is CH₂, CHOH, or C(O), and A is anoptionally substituted phenyl.

In certain embodiments, X is a bond, and A is an optionally substitutedphenyl.

In certain embodiments, p is 2, each X is a bond, and each A is anoptionally substituted phenyl.

In certain embodiments, each B is independently and optionallysubstituted ring selected from:

wherein X₃ is O, S, or NR.

Preferred substituents on B include C1-C4 alkyl, —O—C1-C4 alkyl, CN,halo, COOH, —C(O)NH₂, —C(O)O(C1-C4 alkyl), —C(O)NH(C1-C4 alkyl),—C(O)N(C1-C4 alkyl)₂, or phenyl optionally substituted with up to twosubstituents selected from C1-C4 alkyl, —O—C1-C4 alkyl, CN, halo, COOH,—C(O)NH₂, —C(O)O(C1-C4 alkyl), —C(O)NH(C1-C4 alkyl), —C(O)N(C1-C4alkyl)₂.

In certain embodiments, each B is independently an optionallysubstituted ring selected from ring i, iii, iv, v, or vi. Or, wherein Bis an optionally substituted ring vii.

In some embodiments, B is independently ring x optionally substitutedwith up to two substituents selected from R¹ or phenyl optionallysubstituted with up to two R¹. Preferably, B is phenyl optionallysubstituted with up to two substituents selected from C1-C4 alkyl,—O—C1-C4 alkyl, CN, halo, COOH, —C(O)NH₂, —C(O)O(C1-C4 alkyl),—C(O)NH(C1-C4 alkyl), —C(O)N(C1-C4 alkyl)₂, or phenyl optionallysubstituted with up to two substituents selected from C1-C4 alkyl,—O—C1-C4 alkyl, CN, halo, COOH, —C(O)NH₂, —C(O)O(C1-C4 alkyl),—C(O)NH(C1-C4 alkyl), —C(O)N(C1-C4 alkyl)₂.

Or, each B is an optionally substituted ring selected from ring xi, xii,xiii, or xiv.

In certain embodiments, p is 1. Or, p is 2.

In some embodiments, R is hydrogen.

In some embodiments, Y′ is S. Or, Y′ is O.

In certain embodiments, X is a bond and A is optionally substitutedphenyl. In certain embodiments, A is attached to the carbon atomadjacent to the nitrogen ring atom.

In certain embodiments, A is phenyl optionally substituted with up totwo substituents selected from C1-C4 alkyl, C1-C4 alkoxy, cyano, halo,N-pyrrolidinyl, N-piperidinyl, or methylenedioxy.

In certain embodiments, A is phenyl, methoxyphenyl, dimethoxyphenyl,cyanophenyl, N-pyrrolidinylphenyl, methylenedioxyphenyl, halophenyl,methylphenyl, or dimethylphenyl.

In certain embodiments, A is phenyl, 3-methoxyphenyl, 2-methoxyphenyl,4-chlorophenyl, 4-cyanophenyl, 4-(N-pyrrolidinyl)phenyl, 4-tolyl,3,4-methylenedioxyphenyl, 3-chlorophenyl, 2,4-dimethoxyphenyl,2-chlorophenyl, 4-bromophenyl, or 2,4-dimethylphenyl.

In certain other embodiments, A is C3-C10 cycloaliphatic ring. Exemplaryring include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, oradamantyl. In certain embodiments, A is cyclohexyl or adamantyl.

In certain embodiments the compounds of the present invention have oneof more of the following features:

-   -   a) R is hydrogen;    -   b) Y′ is S;    -   c) A is phenyl optionally substituted with up to two        substituents selected from C1-C4 alkyl, C1-C4 alkoxy, cyano,        halo, N-pyrrolidinyl, N-piperidinyl, or methylenedioxy; and    -   d) B is phenyl optionally substituted with up to two        substituents selected from C1-C4 alkyl, —O—C1-C4 alkyl, CN,        halo, COOH, —C(O)NH₂, —C(O)O(C1-C4 alkyl), —C(O)NH(C1-C4 alkyl),        —C(O)N(C1-C4 alkyl)₂, or phenyl optionally substituted with up        to two substituents selected from C1-C4 alkyl, —O—C1-C4 alkyl,        CN, halo, COOH, —C(O)NH₂, —C(O)O(C1-C4 alkyl), —C(O)NH(C1-C4        alkyl), —C(O)N(C1-C4 alkyl)₂.

In one embodiment, the present invention provides compounds havingformula I-b:

In certain embodiments, p is 1 and X is a bond. In one embodiment, p is1, and X-A is attached to the carbon adjacent to the ring nitrogen atom.Or, p is 1, and X-A is attached to the carbon adjacent to the Y′.

In certain embodiments, p is 1, X is CH₂, CHOH, or C(O), preferably CH₂,and A is an optionally substituted phenyl.

In certain embodiments, X is a bond, and A is an optionally substitutedphenyl.

In certain embodiments, p is 2, each X is a bond, and each A is anoptionally substituted phenyl.

In certain embodiments, said C1-C6 aliphatic is C1-C4 straight orbranched alkylidene. Exemplary alkylidenes include —CH₂—, —CH(Me)—,—C(Me)₂-, —CH(Et)—, —C(Et)₂-, or —CH₂—CH(Me)—.

In certain embodiments, B is selected from optionally substituted C3-C8cycloalkyl, phenyl, piperidyl, or pyrrolidinyl. Preferably, B is phenyl,cyclopentyl, cyclohexyl, or piperidyl, optionally substituted with up totwo R¹ substituents.

In some embodiments, said (C1-C6 aliphatic)-B in formula I-b is selectedfrom:

wherein:

Ak is C1-C6 straight or branched alkylidene;

X₄ is CH₂, O or S;

Ar′ is phenyl optionally substituted with up to two R¹; and

B is optionally substituted with up to two R¹.

In some embodiments, Ak is selected from CH₂, CH(CH₃), C(CH₃)₂, CH(Et),C(Et)₂, CH(n-propyl), CH(i-Pr), CH(n-butyl), CH(but-2-yl), orCH(t-butyl).

In some embodiments, Ar′ is phenyl optionally substituted with halo,C1-C4 alkyl, or O—(C1-C4 alkyl).

In some embodiments, X₄ is S. Or, X₄ is O.

In some embodiments, R is hydrogen.

In one embodiment, the present invention provides compounds of formulaI-f:

wherein:

Y′ is O or S;

X₁ is O, S, or NR;

R^(M) is C1-C6 aliphatic or phenyl, wherein R^(M) is optionallysubstituted with up to two substituents independently selected from R¹,R², or R³;

R^(N) is C1-C6 aliphatic or a 3-7 membered monocyclic, saturated,unsaturated or aromatic ring containing 0-4 heteroatoms in each ring,wherein each said heteroatom is independently selected from N, NH, S, orO;

wherein R^(N) is optionally substituted with up to 4 substituentsindependently selected from R¹, R², R³, R⁴, or R⁵.

In certain embodiments, p is 1 and X is a bond. In one embodiment, p is1, and X-A is attached to the carbon adjacent to the ring nitrogen atom.Or, p is 1, and X-A is attached to the carbon adjacent to the Y′.

In certain embodiments, p is 1, X is CH₂, CHOH, or C(O), preferably CH₂,and A is an optionally substituted phenyl.

In certain embodiments, X is a bond, and A is an optionally substitutedphenyl.

In certain embodiments, p is 2, each X is a bond, and each A is anoptionally substituted phenyl.

In some embodiments, X₁ is NH or N(C1-C4 alkyl). Or, X₁ is O.

In some embodiments, R^(M) is optionally substituted phenyl.

In some embodiments, R^(M) is C1-C6 alkyl, optionally substituted withphenyl. In some embodiments, R^(M) is C1-C4 alkyl.

In some embodiments, R^(N) is optionally substituted C3-C7cycloaliphatic, phenyl, or benzyl.

In some embodiments, R^(N) is C1-C6 aliphatic.

In some embodiments, R is hydrogen.

In one embodiment, the present invention provides a compound of formulaI-g:

wherein:

Y′ is O or S;

R^(P) is C1-C8 aliphatic optionally substituted with up to twosubstituents independently selected from R¹, R², or R³.

In certain embodiments, p is 1 and X is a bond. In one embodiment, p is1, and X-A is attached to the carbon adjacent to the ring nitrogen atom.Or, p is 1, and X-A is attached to the carbon adjacent to the Y′.

In certain embodiments, p is 1, X is CH₂, CHOH, or C(O), preferably CH₂,and A is an optionally substituted phenyl.

In certain embodiments, X is a bond, and A is an optionally substitutedphenyl.

In certain embodiments, p is 2, each X is a bond, and each A is anoptionally substituted phenyl.

In some embodiments, R^(P) is C1-C4 alkyl, optionally substituted withup to two R¹.

In some embodiments, R^(P) is selected from ethyl, n-propyl, i-propyl,n-butyl, but-2-yl, or t-butyl, isoamyl, optionally substituted withhalo, CN, COOH, or CONH₂.

In some embodiments, R is hydrogen.

In certain embodiments, p is 1. Or, p is 2.

In certain embodiments, p is 2, and each A is optionally substitutedphenyl. Or, p is 2, and each A is phenyl.

In certain embodiments, compounds of formula I-g have one or more of thefollowing features:

a) Y′ is S;

b) R is hydrogen;

c) p is 2 and each A is phenyl;

d) R^(P) is isoamyl, t-butyl, ethyl, isopropyl, n-propyl,1-carboxy-prop-3-yl, or 1-carboxy-2-methyl-prop-3-yl.

In one embodiment, the present invention provides compounds of formulaI-h:

wherein:

Y′ is O or S;

X₂ is O, S, or NR;

R^(Q) is C1-C6 aliphatic or phenyl, optionally substituted with up totwo substituents independently selected from R¹, R², or R³.

In certain embodiments, p is 1 and X is a bond. In one embodiment, p is1, and X-A is attached to the carbon adjacent to the ring nitrogen atom.Or, p is 1, and X-A is attached to the carbon adjacent to the Y′.

In certain embodiments, p is 1, X is CH₂, CHOH, or C(O), preferably CH₂,and A is an optionally substituted phenyl.

In certain embodiments, X is a bond, and A is an optionally substitutedphenyl.

In certain embodiments, p is 2, each X is a bond, and each A is anoptionally substituted phenyl.

In some embodiments, X₂ is S. Or, X₂ is O.

In some embodiments, R^(Q) is C1-C4 alkyl, optionally substituted withup to three R¹.

In some embodiments, R^(Q) is phenyl optionally substituted with C1-C4alkyl, or R¹.

According to another preferred embodiment, the methods of the presentinvention employ a compound having formula (IA):

wherein:

-   -   Y′ is O, S, or NR;    -   X is a bond, CH₂, CHOR, C(O)O, C(O), NR, or O;    -   A is aliphatic, aryl, heteroaryl, heterocyclic, or        cycloaliphatic;    -   Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

R is H, R², or R⁶;

wherein each A and B is independently and optionally substituted with upto 4 substituents independently selected from R¹, R², R³, R⁴, or R⁵; and

R¹, R², R³, R⁴, or R⁵ are as defined above for formula (I).

According to one embodiment of formula (IA), Y′ is S.

According to another embodiment of formula (IA), Y′ is 0.

According to another embodiment of formula (IA), Y′ is NR.

In one embodiment, X is a bond, CH₂, NR, or O;

According to another embodiment of formula (IA), X is CH₂. According toanother embodiment of formula (IA), X is CF₂. According to yet anotherembodiment of formula (IA), X is a bond. According to yet anotherembodiment of formula (IA), X is O. According to yet another embodimentof formula (IA), X is NR.

According to another embodiment of formula (IA), A is phenyl or a 5-6membered heteroaryl, preferably phenyl, wherein A is optionallysubstituted with up to 3 substituents selected from R¹, R², R³, or R⁴.

According to one embodiment of formula (IA), Q is B. Alternatively, Q is—(C1-C6)- aliphatic-B. Or, Q is CH(B)₂. According to another embodiment,Q is C(B)₃. Preferably, B is phenyl.

In one embodiment, X is a bond, —CHOR—, or —C(O)—,

Exemplary compounds of formula (IA) are those wherein:

(i) X is a bond, CH₂, or O;

(i) A is optionally substituted phenyl;

(iii) Q is diphenylmethyl.

Exemplary compounds of formula (IA) useful in the methods of the presentinvention are as shown below in Table 1.

According to another embodiment, the methods of the present inventionemploy a compound having formula (IB):

wherein:

-   -   Y′ is O, S, or NR;    -   X is a bond, CH₂, CHOR, C(O), C(O)O, NR, or O;    -   A is aliphatic, aryl, heteroaryl, heterocyclic, or        cycloaliphatic;    -   Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

R is H, R², or R⁶;

wherein each A and B is independently and optionally substituted with upto 4 substituents independently selected from R¹, R², R³, R⁴, or R⁵; and

R¹, R², R³, R⁴, or R⁵ are as defined above for formula (I).

According to one embodiment of formula (IB), Y′ is S.

According to another embodiment of formula (IB), Y′ is 0.

According to another embodiment of formula (IB), Y′ is NR.

According to one embodiment of formula (IB), X is a bond, CH₂, NR, or O,Or, X is a bond or CH₂. According to another embodiment of formula (IB),X is CF₂. According to yet another embodiment of formula (IB), X is abond. According to yet another embodiment of formula (IB), X is O.According to yet another embodiment of formula (IB), X is NR.

According to another embodiment of formula (IB), A is phenyl or a 5-6membered heteroaryl, preferably phenyl, wherein A is optionallysubstituted with up to 3 substituents selected from R¹, R², R³, or R⁴.

According to another embodiment of formula (IB), Q is B. Alternatively,Q is —(C1-C6)- aliphatic-B. Or, Q is CH(B)₂. According to anotherembodiment, Q is C(B)₃. Preferably, B is phenyl.

Preferred compounds of formula IB are those wherein:

(i) X is a bond, CH₂, or O;

(ii) A is optionally substituted phenyl;

(iii) Q is diphenylmethyl.

According to another embodiment, the methods of the present inventionemploy a compound having formula (IC):

wherein:

-   -   Y′ is O, S, or NR;    -   each X is independently a bond, CH₂, —CHOR—, —C(O)O—, —C(O)—,        NR, or O;    -   A is aliphatic, aryl, heteroaryl, heterocyclic, or        cycloaliphatic;    -   Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

R is H, R², or R⁶;

wherein each A and B is independently and optionally substituted with upto 4 substituents independently selected from R¹, R², R³, R⁴, or R⁵; and

R¹, R², R³, R⁴, or R⁵ are as defined above for formula (I).

According to one embodiment of formula (IC), Y′ is S.

According to another embodiment of formula (IC), Y′ is 0.

According to another embodiment of formula (IC), Y′ is NR.

According to another embodiment of formula (IC), X is a bond or CH₂.According to another embodiment of formula (IC), X is CH₂. According toyet another embodiment of formula (IC), X is a bond. Or, X is —CHOR—.Or, X is —C(O)—. According to yet another embodiment of formula (IC), Xis O. According to yet another embodiment of formula (IC), X is NR.

According to another embodiment of formula (IC), A is phenyl or a 5-6membered heteroaryl, preferably phenyl, wherein A is optionallysubstituted with up to 3 substituents selected from R¹, R², R³, or R⁴.

According to another embodiment of formula (IC), Q is B. Alternatively,Q is —(C1-C6)- aliphatic-B. Or, Q is CH(B)₂. According to anotherembodiment, Q is C(B)₃. Preferably, B is phenyl.

Exemplary compounds of formula (IC) are those wherein:

(i) each X is a bond, —CHOR—, or —C(O)—;

(ii) each A is optionally substituted phenyl, (C1-C6)aliphatic, or CF₃;

(iii) Q is optionally substituted phenyl, (C1-C6)aliphatic, ordiphenylmethyl.

According to another embodiment, the present invention provides acompound having formula (II):

or a pharmaceutically acceptable salt thereof;wherein:

X₁ is a bond, O, S, CHOR, C(O), C(O)O, CF₂, CH₂, or NR;

R is H or R²

A₁ is (C2-C10) aliphatic, aryl, heteroaryl, heterocyclic, orcycloaliphatic;

each B₁ is independently selected from 3-7 membered monocyclic,saturated, unsaturated or aromatic ring containing 0-4 heteroatomsselected from N, NH, S, or O;

wherein each A₁ is optionally substituted with up to 4 substituentsindependently selected from R¹, R², R³, R⁴, or R⁵;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(R⁸)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵, R⁶), C(O)N(OR⁶)R⁶, C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)O, C(NOR⁶)R⁵, C(NOR⁵) R⁶,C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵,NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶) R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic, or O-aliphatic;and

R⁸ is an amino capping group.

provided that:

-   -   (i) when both B₁ are simultaneously phenyl and X₁ is CH₂, then A        is not 4-fluoro-phenyl, 4-phenyl-piperidyl, phenyl,        2,4-dichloro-phenyl, 4-methoxy-phenyl, 3,4-dichloro-phenyl,        2,5-dichloro-phenyl, 4-nitro-phenyl, 4-bromo-phenyl,        4-methyl-phenyl, 2-chloro-phenyl, 1-naphthyl,        3-trifluoromethyl-phenyl, 2,3-dichlorophenyl, N-morpholinyl,        4-chloro-phenyl, 3-chloro-phenyl, or 3-nitro-phenyl;    -   (ii) when X₁ is a bond or CH₂, one B₁ is a substituted phenyl        and the other B₁ is cycloaliphatic, then A₁ is not        (C2-C8)aliphatic; and    -   (iii) when X₁ is a bond, then A₁ is not an optionally        substituted 6-membered heteroaryl ring with 1-3 nitrogen ring        atoms.

According to one embodiment of formula (II), X₁ is a bond, O, S, CF₂,CH₂, or NR. X₁ is CH₂, CF₂, or O. Or X₁ is CH₂ or O. In certainembodiments, X₁ is CH₂.

According to another embodiment, A₁ is an optionally substituted C3-C7cycloaliphatic ring. In certain embodiments, A₁ is an optionallysubstituted cyclopropyl, cyclopentyl, or cyclohexyl.

According to another embodiment, A₁ is optionally substituted(C1-C10)aliphatic. In certain embodiments, A₁ is optionally substitutedmethyl, ethyl, propyl, butyl, pentyl, or hexyl.

According to another embodiment, A₁ is optionally substituted C6-C10aryl ring. In certain embodiments, A₁ is optionally substituted phenylor naphthyl.

According to another embodiment, A₁ is optionally substituted C5-C12heteroaryl ring. In certain embodiments, A is selected from optionallysubstituted triazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridinyl,thiadiazolyl, triazolyl, oxadiazolyl, isothiazolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, pyrrolyl, thienyl, furanyl,indolizinyl, indolyl, isoindolyl, benzofuranyl, benzo[b]thienyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, acridinyl, phenazinyl, phenothiazinyl,or phenoxazinyl.

According to another embodiment, A₁ is optionally substituted C₃-C₁₂heterocyclic ring. In certain embodiments, A₁ is selected fromoptionally substituted aziridine, oxirane, thiirane, pyrrolidyl,tetrahydrofuranyl, tetrahydrothienyl, dioxolanyl, pyrrolinyl, pyranyl,pyrazolinyl, pyrazolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl,1,4-dithianyl, thiomorpholinyl, piperazinyl, 3H-indolyl, or indolinyl.

According to another embodiment of formula (II), each B₁ isindependently selected from optionally substituted C6-C10 aryl. Incertain embodiments, each B₁ is independently an optionally substitutedphenyl or naphthyl. Or, each B₁ is an unsubstituted phenyl.

According to another embodiment, each B₁ is independently selected fromoptionally substituted C5-C12 heteroaryl ring. In certain embodiments,each B₁ is independently and optionally substituted C5-C7 heteroarylring. Or, each B₁ is independently selected from optionally substitutedtriazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridinyl, thiadiazolyl,triazolyl, oxadiazolyl, isothiazolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, pyrrolyl, thienyl, furanyl, indolizinyl, indolyl, isoindolyl,benzofuranyl, benzo[b]thienyl, 1H-indazolyl, benzimidazolyl,benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl,phthazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,indenyl, naphthyl, azulinyl, or anthracenyl.

According to another embodiment, each B₁ is independently an optionallysubstituted 3-12 membered heterocyclic ring having up to 4 heteroatomsselected from O, S, or NR. In certain embodiments, each B₁ isindependently selected from optionally substituted aziridine, oxirane,thiirane, pyrrolidyl, tetrahydrofuranyl, tetrahydrothienyl, dioxolanyl,pyrrolinyl, pyranyl, pyrazolinyl, pyrazolidinyl, piperidinyl,1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, piperazinyl,3H-indolyl, or indolinyl.

Embodiments of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and Z in compound offormula (II) are as described above for compound of formula (I).

In certain embodiments, one B₁ is phenyl with up to two R¹ substituents,and the other B₁ is selected from pyrazolyl, pyrrolidinyl, piperidinyl,morpholinyl, thiomorpholinyl, or piperazinyl, optionally substitutedwith up to two R¹ substituents.

In certain embodiments, one B₁ is phenyl, and the other B₁ is selectedfrom 1,2-pyrazol-1-yl, 1-piperidinyl, 2-carboethoxy-1-piperidinyl,4-morpholinyl, 3-carboethoxy-1-piperidinyl, 3-methyl-1-piperidinyl,2-methyl-1-pyrrolidinyl, 3-hydroxymethyl-1-piperidinyl,4-carboethoxy-1-piperidinyl, 4-methyl-1-piperidinyl, 1-pyrrolidinyl,4-(pyrimidin-2-yl)-1-piperazinyl, or 4-hydroxy-piperidinyl.

According to another embodiment, the present invention provides acompound of formula IIA:

wherein:

Y′ is O or S;

B is a 3-8 membered, saturated, monocyclic, ring having 0-4 heteroatomsselected from O, S, or N; and

ring G and B are optionally substituted with up to four substituentsindependently selected from R¹, R², R³, R⁴, or R⁵;

provided that when Y′ is S, and:

a) when B is cyclohexyl, tetrahydrofuran-2-yl, or cyclopropyl, and ringG has 1-3 halo substituents, then ring G has at least one additionalsubstituent other than halo; and

b) when B is tetrahydrofuran-2-yl, then ring G is not phenyl,trifluoromethylphenyl, methoxyphenyl, or tolyl;

c) when B is cyclohexyl, then ring G is not phenyl ortrifluoromethylphenyl.

According to one embodiment, B is tetrahydrofuranyl, piperidyl,morpholinyl, or thiomorpholinyl.

According to another embodiment, B is C3-C8 saturated, carbocyclic,monocyclic ring. Exemplary rings include cyclopropyl, cyclopentyl,cyclohexyl, or cycloheptyl.

According to another embodiment, ring G is phenyl optionally substitutedwith R¹. Preferably, ring G is optionally substituted with up to twosubstituents selected from halo, cyano, C1-C4 alkyl, or O—(C1-C4 alkyl).

In one embodiment, compounds of formula IIA have one or more of thefollowing features:

a) Y′ is S;

b) ring G is halo-substituted phenyl;

c) B is phenyl optionally substituted with halo, cyano, C1-C4 alkyl, orO—(C1-C4 alkyl).

According to another embodiment, the present invention provides acompound of formula IIB:

wherein:

Y′ is S or O;

R is H, R², or R⁶;

R^(B) is C1-C6 aliphatic or a 3-7 membered monocyclic or 8-14 memberedbicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

wherein each of ring G, ring H, and R^(B) is independently andoptionally substituted with up to 4 substituents selected from R¹, R²,R³, R⁴, or R⁵;

provided that when Y is S, and:

a) when R^(B) is hydrogen, and ring G and ring H both have 1-3 halosubstituents, then at least one of ring G and ring H has an additionalsubstituent other than halo;

b) when R^(B) is hydrogen and ring H is unsubstituted phenyl, then ringG is not phenyl or phenyl substituted with methyl, CF₃, —OMe, NO₂, or1-3 halo;

c) when R^(B) is hydrogen and ring H is phenyl with methyl, 1-2 methoxyor 1-2 halo substituents, then ring G is not phenyl substituted with CF₃or 1-2 halo;

d) when R^(B) is methyl and ring H phenyl substituted with butyl, thenring G is not phenyl substituted with methyl, or 1-2 halo; and

e) when R^(B) and ring H are both unsubstituted phenyl, then ring G isnot unsubstituted phenyl, or phenyl substituted with methyl, CF₃, OMe,NO₂, or 1-2 halo.

In certain embodiments, ring G is phenyl optionally substituted with upto two R¹. Exemplary R1 includes C1-C4 alkyl, O—(C1-C4 alkyl), halo, orcyano.

In one embodiment, R^(B) is C₁₋₆ aliphatic optionally substituted withup to 4 substituents selected from R¹, R², R³, R⁴, or R⁵. In certainembodiments, R^(B) is C1-C4 alkyl optionally substituted with up to 2substituents selected from R¹. Exemplary R^(B) include methyl, ethyl,n-propyl, isopropyl, sec-butyl, n-butyl, or t-butyl.

In other embodiments, R^(B) is a 3-7 membered monocyclic saturated,unsaturated or aromatic ring containing 0-4 heteroatoms optionallysubstituted with up to 4 substituents selected from R¹, R², R³, R⁴, orR⁵. In certain embodiments, R^(B) is a 3-7 membered monocyclicsaturated, carbocyclic ring optionally substituted with up to 2substituents selected from R¹. Exemplary rings include cyclopropyl,cyclopentyl, cyclohexyl, or cycloheptyl.

In other embodiments, R^(B) is a 3-7 membered monocyclic saturated,unsaturated or aromatic ring containing 1-3 heteroatoms optionallysubstituted with up to 4 substituents selected from R¹, R², R³, R⁴, orR⁵. In certain embodiments, R^(B) is a 3-7 membered monocyclic saturatedring containing 1-3 heteroatoms optionally substituted with up to 2substituents selected from R¹. Exemplary rings include piperidinyl,morpholinyl, or thiomorpholinyl.

In other embodiments, R^(B) is a 8-14 membered bicyclic or tricyclic,saturated, unsaturated or aromatic ring containing 0-4 heteroatoms ineach ring, wherein each said heteroatom is independently selected fromN, NH, S, or O, optionally substituted with up to 4 substituentsselected from R¹, R², R³, R⁴, or R⁵.

According to another embodiment, the present invention providescompounds of formula IIC:

wherein:

Y′ is O or S;

X₇ is O, S, or NR′;

R′ is hydrogen, R², or R⁶;

R is hydrogen, R², or R⁶;

R^(B) is C1-6 aliphatic or a 3-7 membered monocyclic or 8-14 memberedbicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

R^(C) is C1-C6 aliphatic;

wherein each of ring G, R^(B), and R^(C) is independently and optionallysubstituted with up to 4 substituents selected from R¹, R², R³, R⁴, orR⁵, as defined above.

According to one embodiment, X₇ is O. Or, X₇ is S. Or, X₇ is NR′.

According to one embodiments, R^(C) is C1-C6 alkyl, optionallysubstituted with up to two substituents selected from R¹, R², R³, R⁴, orR⁵. Or, R^(C) is C1-C6 alkyl. Exemplary R^(C) includes methyl, ethyl,isopropyl, n-propyl, n-butyl, sec-butyl, or t-butyl.

According to another embodiment, R^(B) is phenyl optionally substitutedwith up to two R¹ substituents. Or, R^(B) is phenyl.

According to another embodiment, R^(B) is C1-C6 alkyl. Exemplary R^(B)includes methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, ort-butyl.

In one embodiment, compound of formula IIC includes one or more of thefollowing features:

a) ring G is benzyl optionally substituted with one R¹ substituent,preferably halo;

b) Y′ is S and R is hydrogen;

c) R^(C) is C1-C4 alkyl;

d) X₇ is NH or NR′ wherein R′ is C1-C4 alkyl; and

e) R^(B) is C1-C4 alkyl.

According to another embodiment, the present invention provides acompound of formula IID:

wherein:

Y′ is O or S;

R is hydrogen or R²;

X₈ is O, S, or NR′;

R′ is hydrogen, R², or R⁶;

R^(BB) is C₁₋₆ aliphatic or a 3-7 membered monocyclic or 8-14 memberedbicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, S, or O;

wherein each of ring G and R^(BB) is independently and optionallysubstituted with up to 4 substituents selected from R¹, R², R³, R⁴, orR⁵, as defined above.

In certain embodiments, X₈ is O. Or, X₈ is S. Or, X₈ is NR′.

According to another embodiment, R^(BB) is phenyl optionally substitutedwith up to two R¹ substituents. Or, R^(BB) is phenyl.

According to another embodiment, R^(BB) is C1-C6 alkyl. Exemplary R^(BB)includes methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, ort-butyl.

According to another embodiment, R^(BB) is optionally substituted C3-C8cycloalkyl, e.g., cyclopropyl, cyclopentyl, or cyclohexyl.

According to another embodiment, R^(BB) is optionally substitutedbenzyl.

In one embodiment, compounds of formula IID have one or more of thefollowing features:

a) Y′ is S and R is hydrogen;

b) each ring G is unsubstituted phenyl;

d) X₈ is NR′, and R′ is hydrogen or C1-C4 alkyl; and

e) R^(BB) is C1-C4 alkyl, benzyl, cyclopentyl, or cyclohexyl.

According to another preferred embodiment, the present inventionprovides a compound having formula (III):

or a pharmaceutically acceptable salt thereof;wherein:

X₁ is a bond, O, S, CHOR, C(O), C(O)O, CF₂, CH₂, or NR;

R is H or R²

A₁ is (C2-C10) aliphatic, aryl, heteroaryl, heterocyclic, orcycloaliphatic;

each B₁ is independently selected from 3-7 membered monocyclic,saturated, unsaturated or aromatic ring containing 0-4 heteroatomsselected from N, NH, S, or O;

wherein each A₁ is optionally substituted with up to 4 substituentsindependently selected from R¹, R², R³, R⁴, or R⁵;

R¹ is R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(O)R⁶, C(O)N(OR⁵)R⁶,C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵) R⁶, C(NOR⁵)R⁵,N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵, NR⁶C(O)OR⁶,NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵, NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶,NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶, NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶,NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂, NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂,NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂, N(OR⁶)R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, N-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, COOH, C(O)O(-aliphatic, or O-aliphatic; and

R⁸ is an amino capping group;

provided that:

-   -   (i) when X₁ is a bond, one B₁ is phenyl and the other B₁ is        N-piperidyl, then A is not:

and

-   -   (ii) when X₁ is a bond, then A₁ is not an optionally substituted        6-membered heteroaryl ring with 1-3 nitrogen ring atoms.

In certain embodiments, According to another embodiment of formula(III), X₁ is CH₂, CF₂, or O.

In another embodiment, X₁ is a bond, O, S, CF₂, CH₂, or NR. Or, X₁ isCH₂ or O. Or, X₁ is CH₂.

According to another embodiment of formula (III), A₁ is an optionallysubstituted C3-C7 cycloaliphatic ring. In certain embodiments, A₁ is anoptionally substituted cyclopropyl, cyclopentyl, or cyclohexyl.

According to another embodiment of formula (III), A₁ is optionallysubstituted (C1-C10)aliphatic. In certain embodiments, A₁ is optionallysubstituted methyl, ethyl, propyl, butyl, pentyl, or hexyl.

According to another embodiment of formula (III), A₁ is optionallysubstituted C6-C10 aryl ring. In certain embodiments, A₁ is optionallysubstituted phenyl or naphthyl.

According to another embodiment of formula (III), A₁ is optionallysubstituted C5-C12 heteroaryl ring. In certain embodiments, A isselected from optionally substituted triazinyl, pyrazinyl, pyrimidinyl,pyridazinyl, pyridinyl, thiadiazolyl, triazolyl, oxadiazolyl,isothiazolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, pyrrolyl,thienyl, furanyl, indolizinyl, indolyl, isoindolyl, benzofuranyl,benzo[b]thienyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, acridinyl, phenazinyl,phenothiazinyl, or phenoxazinyl.

According to another embodiment of formula (III), A₁ is optionallysubstituted C3-C12 heterocyclic ring. In certain embodiments, A₁ isselected from optionally substituted aziridine, oxirane, thiirane,pyrrolidyl, tetrahydrofuranyl, tetrahydrothienyl, dioxolanyl,pyrrolinyl, pyranyl, pyrazolinyl, pyrazolidinyl, piperidinyl,1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, piperazinyl,3H-indolyl, or indolinyl.

According to another embodiment of formula (III), each B₁ isindependently selected from optionally substituted C6-C10 aryl. Incertain embodiments, each B₁ is independently an optionally substitutedphenyl or naphthyl. Or, each B₁ is an unsubstituted phenyl.

According to another embodiment of formula (III), each B₁ isindependently selected from optionally substituted C5-C12 heteroaryl. Incertain embodiments, each B₁ is independently and optionally substitutedC5-C7 heteroaryl. Or, each B₁ is independently selected from optionallysubstituted triazinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyridinyl,thiadiazolyl, triazolyl, oxadiazolyl, isothiazolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, pyrrolyl, thienyl, furanyl,indolizinyl, indolyl, isoindolyl, benzofuranyl, benzo[b]thienyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, indenyl, naphthyl, azulinyl, oranthracenyl.

According to another embodiment of formula (III), each B₁ isindependently an optionally substituted 3-12 membered heterocyclic ringhaving up to 4 heteroatoms selected from O, S, or NR. In certainembodiments, each B₁ is independently selected from optionallysubstituted aziridine, oxirane, thiirane, pyrrolidyl, tetrahydrofuranyl,tetrahydrothienyl, dioxolanyl, pyrrolinyl, pyranyl, pyrazolinyl,pyrazolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,thiomorpholinyl, piperazinyl, 3H-indolyl, or indolinyl.

Exemplary embodiments of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and Z incompounds of formula (III) are as described above for compound offormula (I).

According to another embodiment, the present invention providescompounds of formula IV:

wherein:

B is selected from:

wherein:

Y is O or S; and

X₃ is O or S.

According to one embodiment, X₃ is O. Or, X₃ is S.

According to one embodiment, B is structure (i) above. Or, B isstructure (ii) above.

According to another embodiment, R is hydrogen.

According to another embodiment, Y′ is S. Or, Y′ is O.

According to another embodiment, the present invention providescompounds of formula V:

wherein:

Y′ is O or S;

B is selected from:

wherein:

Ak is C1-C6 alkylidene;

X₄ is CH₂, O or S;

Ar′ is phenyl optionally substituted with up to two R¹; and

B is optionally substituted with up to two R′.

In some embodiments, Ak is selected from CH₂, CH(CH₃), C(CH₃)₂, CH(Et),C(Et)₂, CH(n-propyl), CH(i-Pr), CH(n-butyl), CH(but-2-yl), orCH(t-butyl).

In some embodiments, Ar′ is phenyl optionally substituted with halo,C1-C4 alkyl, or O—(C1-C4 alkyl).

In some embodiments, X₄ is CH₂. X₄ is S. Or, X₄ is O.

In some embodiments, R is hydrogen.

In certain embodiments, p is 2, X is a bond, and each A is optionallysubstituted phenyl.

In other embodiments, the compounds have one or more of the followingfeatures:

a) Y′ is S;

b) R is hydrogen;

c) p is 2, X is a bond, and each A is phenyl;

d) B is ring (iii) above, wherein Ak is CH(CH₃) and Ar′ is phenyloptionally substituted with halo, C1-C4 alkyl, or O—(C1-C4 alkyl).

According to another embodiment, the present invention provides acompound of formula VI:

Y′ is O or S;

R is hydrogen or R²;

B is phenyl, 3-7 membered, monocyclic, saturated, carbocyclic ring, or3-10 membered saturated or unsaturated, monocyclic or bicyclicheterocyclic ring having up to 4 heteroatoms selected from O, S, or N,or 5-10 membered monocyclic or bicyclic heteroaryl ring having up to 4heteroatoms selected from O, S, or N;

wherein each ring G₁, G₂, and B is independently substituted with up to4 substituents selected from R¹, R², R³, R⁴, or R⁵;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(R⁸)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁵, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶, C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵)R⁶,C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵,NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶)R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic, or O-aliphatic;and

R⁸ is an amino capping group.

In certain embodiments of formula VI, when R is hydrogen, then thefollowing compounds are excluded:

a) B is not quinolin-2-yl or 1,2-dihydro-2-oxo-quinolin-4-yl;

b) when G₁ and G₂ both are phenyl, and Y′ is S, then B is not1,4-benzodioxin-2-yl, cyclopropyl, cyclohexyl, thien-2-yl,1H-thieno[2,3-c]pyrazol-1-phenyl-3-methyl-5-yl, 5-methyl-thien-3-yl,2,5-dichloro-thien-3-yl, 2-phenyl-quinolin-4-yl, furan-2-yl,thien-5-(4,5-diphenyl-2-thiazolyl-carboxamide)-2-yl,benzo[b]thiophen-2-yl,pyridin-2-(4,5-diphenyl-2-thiazolyl-carboxamide)-6-yl,5-nitro-thien-2-yl, 3-chloro-benzo[b]thiophen-2-yl, 4H-1-benzopyran-3-ylor 2H-1-benzopyran-3,4-dihydro-3-oxo-4-yl, 4H-1-benzopyran-3-yl or2H-1-benzopyran-3,4-dihydro-3-oxo-4-yl;c) when G₁ and G₂ both are phenyl, and Y′ is O, then B is not1,2-dihydro-2-oxo-quinolin-4-yl or 3,4-dihydro-3-phenyl-phthalazin-1-ylor thien-2-yl;d) the following compounds are excluded:

y′ G₁ G₂ B S Ph 4-Me—Ph 2-Cl-thien-5-yl or 2,5-dichloro-thien-3-yl S Phor 3,4- 3,4-dimethyl pyridin-3-yl dimethyl phenyl phenyl or Ph S3,4-dimethyl phenyl Ph pyridin-4-yl S 4-Cl—Ph Ph thien-2-yl S3,4-dimethyl phenyl Ph pyridin-4-yl S Ph 4-Me-phenyl thien-2-yl orbenzothiazol-2-yl S 4-NO₂—Ph or Ph or 4-OMe—Ph or thien-2-yl or 4-Me—Ph2,4-dimethylphenyl furan-2-yl S 4-OMe—Ph or 4-NO₂—Ph furan-2-yl or2,4-dimethylphenyl thien-2-yl S 4-OMe—Ph or Ph 4-OMe—Ph furan-2-yl orthien-2-yl S Ph 4-Me—Ph furan-2-yl S Ph 4-Me—Ph 5-nitro-thien-2-yl S4-Me—Ph 4-Me—Ph 2-chloro-pyridin-3-yl S Ph 4-Me—Ph 3-chloro-benzo[b]thiophen-2-yl S 4-Me—Ph or Ph 4-NO₂—Ph thien-2-yl or furan-2-yl S2,4-dimethylphenyl 4-NO₂—Ph pyridin-3-yl or thien-2-yl S 4-Cl—Ph4-NO₂—Ph thien-2-yl S Ph or 2-OMe—Ph or 2,4-dimethoxy-phenyl1H-indol-2-yl. 3-OMe—Ph or 4-OMe—Phd) when Y′ is S, G₁ and G₂ are both phenyl, then B is not

In one embodiment, G₁ and G₂ are both phenyl. Or, each is independentlyand optionally substituted with up to two substituents selected fromhalo, or C1-C4 alkyl.

In certain embodiments of formula VI, B is phenyl optionally substitutedwith up to two substituents selected from halo, C1-C4 alkyl, O—(C1-C4alkyl), COOH, COO(C1-C4 alkyl), or cyano.

In other embodiments, B is 3,4-dichlorophenyl, 4-chlorophenyl,4-methoxyphenyl, 4-methylphenyl, 2,6-difluorophenyl, 2-methylphenyl,3-methylphenyl, phenyl-2-carboxylic acid, 2-chlorophenyl, 4-cyanophenyl,or 3-methoxyphenyl.

In another embodiment, B is 3-7 membered, monocyclic, saturated,carbocyclic ring. Exemplary rings include cyclopropyl, cyclopentyl,cyclohexyl, or cycloheptyl.

In another embodiment, B is a 3-10 membered saturated or unsaturated,monocyclic or bicyclic heterocyclic ring having up to 4 heteroatomsselected from O, S, or N. Exemplary rings include tetrahydrofuranyl,thienyl, or pyrrolyl.

In another embodiment, B is a 5-10 membered monocyclic or bicyclicheteroaryl ring having up to 4 heteroatoms selected from O, S, or N.

According to another embodiment, the present invention provides acompound of formula VII:

wherein:

Y′ is O or S;

R is hydrogen or R²;

R^(Ak) is C1-C6 aliphatic, optionally substitute with up to 3substituents independently selected from R¹, R², or R³;

B is phenyl, 3-7 membered, monocyclic, saturated, carbocyclic ring, or3-10 membered saturated or unsaturated, monocyclic or bicyclicheterocyclic ring having up to 4 heteroatoms selected from O, S, or N,or 5-10 membered monocyclic or bicyclic heteroaryl ring having up to 4heteroatoms selected from O, S, or N;

wherein each ring G₁, G₂, and B is independently substituted with up to4 substituents selected from R¹, R², R³, R⁴, or R⁵;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(R⁸)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶, C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵)R⁶,C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶); NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵,NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O) NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂,NR⁵C(O)NR⁵R⁶, NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶,NR⁶SO₂N(R⁶)₂, NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶,NR⁵SO₂N(R⁵)₂, N(OR⁶) R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic, or O-aliphatic;and

R⁸ is an amino-capping group.

In one embodiment of formula VII, when each of G₁, G₂, and B isunsubstituted phenyl, and R is hydrogen, then B is not3,4,5-trimethoxyphenyl.

In one embodiment, R^(Ak) is C1-C6 alkyl. Exemplary R^(Ak) includemethyl, ethyl, isopropyl, n-propyl, sec-butyl, n-butyl, or t-butyl.

In one embodiment, B is optionally substituted phenyl.

In another embodiment, B is 3-7 membered, monocyclic, saturated,carbocyclic ring. Exemplary rings include cyclopropyl, cyclopentyl,cyclohexyl, or cycloheptyl.

In another embodiment, B is a 3-10 membered saturated or unsaturated,monocyclic or bicyclic heterocyclic ring having up to 4 heteroatomsselected from O, S, or N. Exemplary rings include tetrahydrofuranyl,tetrahydrothiophenyl, pyrrolidinyl, or piperazinyl.

In another embodiment, B is a 5-10 membered monocyclic or bicyclicheteroaryl ring having up to 4 heteroatoms selected from O, S, or N.

According to another embodiment, the present invention provides acompound having formula I′:

or a pharmaceutically acceptable salt thereof;wherein:

Y′ is O, S, or NR; R is H, R², or R⁶;

C is a phenyl or 5-8 membered cycloaliphatic ring;

Q is selected from:

each B is independently selected from 3-7 membered monocyclic or 8-14membered bicyclic or tricyclic, saturated, unsaturated or aromatic ringcontaining 0-4 heteroatoms in each ring, wherein each said heteroatom isindependently selected from N, NH, S, or O;

wherein each A, B, and C is independently and optionally substitutedwith up to 4 substituents independently selected from R¹, R², R³, R⁴, orR⁵;

R^(L) is —OR^(A), —SR^(A), or —N(R^(AB))₂;

each R^(A) is independently hydrogen, C1-C6 aliphatic, or a 3-7 memberedcarbocyclic or heterocyclic ring, saturated or unsaturated ring, havingup to 3 heteroatoms selected from O, N, or S, wherein each R^(A) isoptionally substituted with up to 3 substituents independently selectedfrom R¹, R⁴ or R⁷,

each R^(AB) is independently hydrogen or C1-C6 aliphatic optionallysubstituted with up to 3 substituents independently selected from R¹, R⁴or R⁷;

wherein up to two methylene units in R^(A) or R^(AB) are optionallyreplaced with —CO—, —CS—, —COCO—, —CONR—, —CO₂—, —OCO—, —NRCO₂ ⁻, —O—,—NRCONR—, —OCONR—, —NRCO—, —S—, —SO, —SO₂—, —NR—, —SO₂NR—, NRSO₂—, or—NRSO₂NR; or

two R^(AB), taken together with the nitrogen atom, is a 3-7 memberedheterocyclic or heteroaryl ring containing up to 4 heteroatoms selectedfrom O, N, or S, wherein said ring is optionally substituted with up to2 substituents selected from oxo or (C₁₋₄aliphatic)_(p)-Y;

R^(N) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

each of X₁ and X₂ is independently selected from O, S, or NR;

R^(N) is C1-C6 aliphatic or phenyl, wherein R^(N) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R^(P) is C1-C6 aliphatic, optionally substituted with up to twosubstituents selected from R¹, R², R³, or R⁴;

R^(Q) is C1-C6 aliphatic or aryl, wherein R^(Q) is optionallysubstituted with up to two substituents selected from R¹, R², R³, or R⁴;

R¹ is oxo, R⁶ or ((C1-C4)aliphatic)_(n)-Y;

n is 0 or 1;

Y is halo, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶,N(R⁶)₂, NR⁶R⁸, N(R⁸)², COOH, COOR⁶ or OR⁶; or

two R¹ on adjacent ring atoms, taken together, form 1,2-methylenedioxyor 1,2-ethylenedioxy;

R² is aliphatic, wherein each R² optionally comprises up to 2substituents independently selected from R¹, R⁴, or R⁵;

R³ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ringoptionally comprising up to 3 substituents, independently selected fromR¹, R², R⁴ or R⁵;

R⁴ is OR⁵, OR⁶, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶, OC(O)OR⁵, OC(O)N(R⁶)₂,OC(O)N(R⁵)₂, OC(O)N(R⁶R⁵), SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶, SO₂R⁵,SO₂N(R⁶)₂, SO₂N(R⁵)₂, SO₂NR⁵R⁶, SO₃R⁶, SO₃R⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶,C(O)OR⁶, C(O)N(R⁶)₂, C(O)N(R⁵)₂, C(O)N(R⁵R⁶), C(O)N(OR⁶)R⁶,C(O)N(OR⁵)R⁶, C(O)N(OR⁶)R⁵, C(O)N(OR⁵)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, C(NOR⁵)R⁶, C(NOR⁵)R⁵, N(R⁶)₂, N(R⁵)₂, N(R⁵R⁶), NR⁵C(O)R⁵, NR⁶C(O)R⁶, NR⁶C(O)R⁵,NR⁵C(O)R⁶, NR⁶C(O)OR⁶, NR⁵C(O)OR⁶, NR⁶C(O)OR⁵, NR⁵C(O)OR⁵,NR⁶C(O)N(R⁶)₂, NR⁶C(O)NR⁵R⁶, NR⁶C(O)N(R⁵)₂, NR⁵C(O)N(R⁶)₂, NR⁵C(O)NR⁵R⁶,NR⁵C(O)N(R⁵)₂, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁵SO₂R⁵, NR⁵SO₂R⁶, NR⁶SO₂N(R⁶)₂,NR⁶SO₂NR⁵R⁶, NR⁶SO₂N(R⁵)₂, NR⁵SO₂N(R⁶)₂, NR⁵SO₂NR⁵R⁶, NR⁵SO₂N(R⁵)₂,N(OR⁶)R⁶, N(OR⁶)R⁵, N(OR⁵)R⁵, or N(OR⁵)R⁶;

R⁵ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring,optionally comprising up to 3 R¹ substituents;

R⁶ is H or aliphatic, wherein R⁶ optionally comprises a R⁷ substituent;

R⁷ is a cycloaliphatic, aryl, heterocyclic, or heteroaryl ring, and eachR⁷ optionally comprises up to 2 substituents independently chosen fromH, (C₁-C₆)-straight or branched alkyl, (C₂-C₆) straight or branchedalkenyl or alkynyl, 1,2-methylenedioxy, 1,2-ethylenedioxy, or(CH₂)_(n)—Z;

Z is selected from halo, CN, NO₂, CF₃, OCF₃, OH, S-aliphatic,S(O)-aliphatic, SO₂-aliphatic, NH₂, NH-aliphatic, N(aliphatic)₂,N(aliphatic)R⁸, NHR⁸, N(R⁸)₂, COOH, C(O)O(-aliphatic, or O-aliphatic;and

R⁸ is an amino-capping group.

In one embodiment, Y′ is O. Or, Y′ is S.

In another embodiment, R is hydrogen.

In one embodiment, Q in formula I′ is B (structure (a)). Preferred Binclude optionally substituted phenyl, or C3-C8 cycloaliphatic. In oneembodiment, B in formula I′ is phenyl or C3-C8 cycloalkyl optionallysubstituted with up to two substituents selected from R¹, R², or phenyloptionally substituted with up to two substituents selected from R¹ orR².

In another embodiment, Q in formula I′ is —(C1-C6 aliphatic)-B(structure (b)).

In certain embodiments, said C1-C6 aliphatic is C1-C4 straight orbranched alkylidene. Exemplary alkylidenes include —CH₂—, —CH(Me)—,—C(Me)₂-, —CH(Et)-, —C(Et)₂-, or —CH₂—CH(Me)-.

In certain embodiments, B is selected from optionally substituted C3-C8cycloalkyl, phenyl, piperidyl, or pyrrolidinyl. Preferably, B is phenyl,cyclopentyl, cyclohexyl, or piperidyl, optionally substituted with up totwo R¹ or R² substituents.

In one embodiment, ring C is phenyl optionally substituted with up totwo R¹. Or ring C is cyclohexenyl optionally substituted with up to twoR¹.

According to a preferred embodiment, R¹ is 1,2-methylene dioxy, or1,2-ethylenedioxy.

According to another preferred embodiment, R¹ is R⁶, wherein R⁶ isstraight chain or branched (C1-C6)alkyl or (C2-C6) alkenyl or alkynyl,optionally substituted with R⁷.

According to another preferred embodiment, R¹ is (C1-C4aliphatic)_(n)-Y, wherein n is 0 or 1, and Y is halo, CN, NO₂, CF₃,OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶, NH₂, NHR⁶, N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ orOR⁶;

According to another preferred embodiment, R¹ is selected from halo,CF₃, NH₂, NH(C1-C4 alkyl), NHC(O)CH₃, OH, O(C1-C4 alkyl), OPh, O-benzyl,S—(C1-C4 alkyl), C1-C4 aliphatic, CN, methylenedioxy, ethylenedixoy,SO₂NH(C1-C4 alkyl), or SO₂N(C1-C4 alkyl)₂.

According to another more preferred embodiment, R¹ is selected frommethyl, n-propyl, propyl, t-butyl, halo, CF₃, NH₂, NH(CH₃), NHC(O)CH₃,OH, OCH₃, OPh, O-benzyl, S—(C₂H₅), S—CH₃, NO₂, CN, methylenedioxy,SO₂NH(n-propyl), or SO₂N(n-propyl)₂.

According to a preferred embodiment, R² is a straight chain or branched(C1-C6)alkyl or (C2-C6) alkenyl or alkynyl, optionally substituted withR¹, R⁴, or R⁵. More preferably, R² is a straight chain or branched(C1-C4)alkyl or (C2-C4) alkenyl or alkynyl, optionally substituted withR¹, R⁴, or R⁵.

In formula I′, other embodiments of B, R^(L), R^(M), R^(N), R^(P),R^(Q), X₁, X₂, and R are as described above for formula I.

Exemplary compounds of the present invention are shown below in Table 1.

TABLE 1 Cmpd # Compound 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

According to an alternative embodiment, preferred compounds of thepresent invention are those that measurably increase the activity of anABC-transporter or of a fragment thereof, and preferably CFTR activity.

According to another alternative embodiment, preferred compounds of thepresent invention are those that measurably decrease the activity of anABC-transporter or of a fragment thereof.

One of skill in the art would be well aware of techniques and assaysuseful in measuring the increase or decrease of activity of anABC-transporter or of a fragment thereof.

According to an alternative preferred embodiment, the present inventionprovides a method of modulating CFTR activity in a cell membrane of amammal in need thereof, comprising the step of administering to saidmammal a composition comprising a compound of the present invention asdefined above.

The preferred embodiments of compound of formula (I) useful inpotentiating the activity of CFTR include the preferred embodiments ofthe present invention described above.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of the present invention. The term “functional ABC transporter”as used herein means an ABC transporter that is capable of transportactivity.

According to a preferred embodiment, said functional ABC transporter isCFTR.

The preferred embodiments of compounds of formula (I) useful inincreasing the number of functional ABC transporters include preferredembodiments of the compounds of the present invention as describedabove.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools or probes in biological assays.

The present invention includes within its scope pharmaceuticallyacceptable prodrugs of the compounds of the present invention. A“pharmaceutically acceptable prodrug” means any pharmaceuticallyacceptable salt, ester, salt of an ester, or other derivative of acompound of the present invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention or an active metabolite or residue thereof. Preferredprodrugs are those that increase the bioavailability of the compounds ofthis invention when such compounds are administered to a mammal or whichenhance delivery of the parent compound to a biological compartmentrelative to the parent species.

The compounds of the present invention may be readily prepared usingmethods known in the art. One such synthetic route is illustrated inScheme 1 below:

2-Aminothiazoles

The appropriate bromoketone was dissolved in a minimum of ethanol ormethanol with 1.1 equivalents of thiourea. The reaction mixture wasstirred overnight at room temperature, evaporated to dryness, and thendissolved in either dichloromethane or ethyl acetate. The reactionmixture was then extracted with 1M sodium hydroxide followed by asaturated aqueous sodium chloride solution. The organic layer was thenseparated, dried over sodium sulfate, and evaporated to dryness to yieldthe desired 2-aminothiazole.

Step 2:

Amides.

(a) If the appropriate acid chloride was commercially available, it wasadded to one equivalent of the appropriate amine in minimum of pyridine.The reaction mixture was allowed to stir overnight at room temperature.The reaction mixture was then filtered and evaporated to dryness. Thecrude product was purified by reverse phase preparative liquidchromatography.

(b) Alternatively, the acid chloride was added to one equivalent of theappropriate amine in minimum of 1,4-dioxane containing an excess oftriethylamine. The reaction mixture was then either allowed to stirovernight at room temperature or subjected to microwave irradiation for5 minutes at 200° C. The crude product was then filtered, evaporated todryness, dissolved in a minimum of dimethylsulfoxide and then purifiedby reverse phase preparative liquid chromatography.

(c) If the appropriate acid chloride was not commercially available theappropriate carboxylic acid was added to a solution containing oneequivalent of the appropriate amine in a minimum of pyridine.O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 1.4 eq.) is added, and the reaction isstirred overnight. The crude product was purified by reverse phasepreparative liquid chromatography.

(d) If the appropriate acid chloride was not commercially available theappropriate carboxylic acid was added to a solution containing oneequivalent of the appropriate amine in a minimum ofN,N-dimethylformamide with an excess of triethylamine.O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 1.4 eq.) is added, and the reaction isstirred overnight. The crude product was purified by reverse phasepreparative liquid chromatography.

One of skill in the art will recognize that the above two syntheticroutes are generic and can be readily exploited for any embodiment ofcompound formula (I).

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium and N⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternization ofany basic nitrogen-containing groups of the compounds disclosed herein.Water or oil-soluble or dispersible products may be obtained by suchquaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe modulator can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Depending upon the particular condition, or disease, to be treated orprevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may also be present inthe compositions of this invention. As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated.”

According to an alternative embodiment, the present invention provides amethod of treating a ABC transporter mediated disease in a mammal,comprising the step of administering to said mammal a compositioncomprising a compound of the present invention, or a preferredembodiment thereof as set forth above.

According to a preferred embodiment, the ABC transporter mediateddisease is selected from immunodeficiency disorder, inflammatorydisease, allergic disease, autoimmune disease, destructive bonedisorder, proliferative disorder, infectious disease or viral disease.

In certain preferred embodiments, the present invention provides amethod of treating cystic fibrosis, hereditary emphysema, hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, Type-1 hereditary angioedema, lipid processingdeficiencies, such as Familial hypercholesterolemia, Type-1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, hereditary emphysema, congenital hyperthyroidism,osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker disease, secretory diarrhea, polycystic kidneydisease, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome, comprising the step of administering to saidmammal an effective amount of a composition comprising a compound of thepresent invention, or a preferred embodiment thereof as set forth above.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of the present invention.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of any one of the above diseases.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any routeadministration effective for treating or lessening the severity of anyone of the above diseases.

In one embodiment, the compounds of the present invention are useful intreating cystic fibrosis.

According to a more preferred embodiment, the disease so treated isselected from Tangier's disease, stargardt disease 1, age relatedmacular dystrophy 2, retinintis pigmentosa, bare lymphocyte syndrome,PFIC-3, anemia, progressive intrahepatic cholestasis-2, Dublin-Johnsonsyndrome, Pseudoxanthoma elasticum, cystic fibrosis, familial persistenthyperinsulinemic hypoglycemia of infancy, adrenolecukodystrophy,sitosterolemia, chronic obstructive pulmonary disease, asthma,disseminated bronchiectasis, chronic pancreatitis, male infertility,emphysema, or pneumonia.

According to another more preferred embodiment, the ABC transportermediated disease is secretory diarrhea, or polycystic kidney disease ina mammal.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis or secretory diarrheacomprising the step of administering to said mammal a compositioncomprising the step of administering to said mammal a compositioncomprising a compound of the present invention, or a preferredembodiment thereof as set forth above. Most preferably, said disease iscystic fibrosis.

According to another embodiment, the present invention provides a methodof modulating activity of an anion channel in vitro or in vivo,comprising the step of contacting said channel with a compound of thepresent invention. Preferably, said anion channel is a chloride channelor a bicarbonate channel. More preferably, said anion channel is achloride channel.

According to yet another embodiment, the present invention provides amethod of treating an anion channel mediated disease in a mammal,comprising the step of administering to said mammal a compositioncomprising a compound according to the present invention.

According to another embodiment, the present invention provides apharmaceutical composition comprising:

(i) a compound of the present invention as described above;

(ii) a pharmaceutically acceptable carrier; and

(iii) an additional agent selected from a mucolytic agent,bronchodialator, an antibiotic, an anti-infective agent, ananti-inflammatory agent, a CFTRmodulator, or a nutritional agent.

Preferred embodiments of compounds the present invention in the abovepharmaceutical composition are those as described above.

According to another embodiment, the present invention provides a kitfor use in measuring the activity of a ABC transporter or a fragmentthereof in a biological sample in vitro or in vivo, comprising:

(i) a composition comprising a compound of the present invention; and

(ii) instructions for:

-   -   a) contacting the composition with the biological sample;    -   b) measuring activity of said ABC transporter or a fragment        thereof.

According to a preferred embodiment, the kit is useful in measuring theactivity of CFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential.

Means for measuring the voltage potential across a membrane in thebiological sample may employ any of the known methods in the art, suchas optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

Preferred ABC transporters in the kit of the present invention includeCFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1

General Procedure:

The appropriate bromoketone was dissolved in a minimum of ethanol ormethanol with 1.1 equivalents of thiourea. The reaction mixture wasstirred overnight at room temperature, evaporated to dryness, and thendissolved in either dichloromethane or ethyl acetate. The reactionmixture was then extracted with 1 M sodium hydroxide followed by asaturated aqueous solution of sodium chloride. The organic layer wasthen separated, dried over sodium sulfate, and evaporated to dryness toyield the desired 2-aminothiazole.

EXAMPLE 2

General Procedure:

One equivalent of the appropriate carboxylic acid and one equivalent ofthe appropriate amine were dissolved in N,N-dimethylformamide (DMF)containing triethylamine (3 equivalents).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) was added and the solution was allowed tostir. The crude product was purified by reverse-phase preparative liquidchromatography to yield the pure product.

EXAMPLE 3

General Procedure:

The appropriate acid chloride was added to one equivalent of theappropriate amine in minimum of pyridine. The reaction mixture wasallowed to stir overnight at room temperature. The reaction mixture wasthen filtered and evaporated to dryness. The crude product was purifiedby reverse-phase preparative liquid chromatography. Alternatively, theacid chloride was added to one equivalent of the appropriate amine inminimum of 1,4-dioxane containing an excess of triethylamine. Thereaction mixture was then either allowed to stir overnight at roomtemperature or subjected to microwave irradiation for 5 minutes at 200°C. The crude product was then filtered, evaporated to dryness, dissolvedin a minimum of dimethylsulfoxide and then purified by reverse-phasepreparative liquid chromatography.

EXAMPLE 4

wherein W is a group as described in the compounds of the presentinvention.

General Procedure:

One equivalent of the halide was dissolved in a minimum of the alcohol.The reaction vessel was sealed and then subjected to microwaveirradiation for 15 minutes at 125° C. The crude mixture was evaporatedto dryness, dissolved in 1 mL of dimethylsulfoxide and purified byreverse-phase preparative liquid chromatography.

EXAMPLE 5

wherein W is a group as described in the compounds of the presentinvention.

General Procedure:

One equivalent of the halide was dissolved in a minimum ofN,N-dimethylformamide (DMF) containing 20 equivalents of amine. Thereaction vessel was sealed and then subjected to microwave irradiationfor 5 minutes at 80° C. The crude mixture was evaporated to dryness andpurified by reverse-phase preparative liquid chromatography.

a) 4-Fluoro-1-methoxy-2-nitro-benzene

A solution of methyl iodide (127.8 g, 0.9004 mol) in acetonitrile (100mL) was slowly added to a solution of 5-fluoro-2-nitrophenol (94.2 g,0.600 mol) and potassium carbonate (207 g, 1.50 mol) in acetonitrile(450 mL). The mixture was heated to reflux for 15 hours. The mixture wasallowed to cool, filtered, and washed twice with dichloromethane (100mL). The combined filtrated was evaporated to dryness to give thedesired product (96 g, 0.56 mol, 93%), which was used directly in thenext step.

b) 5-Fluoro-2-methoxy-phenylamine

A solution of 4-fluoro-1-methoxy-2-nitro-benzene (85 g, 0.50 mol) inmethanol (300 mL) containing palladium on carbon (10%, 8 g) was stirredfor 15 hours under an atmosphere of hydrogen. The catalyst was filteredand the filtrate was evaporated to dryness to give the crude product(61.5 g, 0.436 mol, 87%), which was used directly in the next step.

c) 2-Chloro-3-(5-fluoro-2-methoxy-phenyl)-propionaldehyde

A solution of sodium nitrite (36 g, 0.51 mole) in water (50 mL) wasslowly added to a solution of 5-fluoro-2-methoxy-phenylamine (61.5 g,0.48 mol) in hydrochloric acid (20% aqueous solution, 115 mL) at 0° C.After stirring for 10 minutes, a cooled (0° C.) solution of acrolein (50mL, 0.75 mol) in acetone (100 mL) containing calcium oxide (0.56 g,0.010 mol) was added slowly to the reaction mixture. This was thenfollowed by a solution of cuprous chloride (5 g, 0.05 mol) in acetone(100 mL) containing hydrochloric acid (20% aqueous solution, 10 mL). Themixture was stirred at 0 to 30° C. for 3 hours, and then extracted threetimes with dichloromethane (300 mL). The combined organic layers werewashed with a saturated aqueous solution of sodium bicarbonate, asaturated aqueous solution of sodium chloride, dried over sodiumsulfate, filtered, and evaporated to dryness to give a black viscousoil. The crude product was passed through a short silica column to givethe crude product, which was used directly in the next step.

d) 5-(5-Fluoro-2-methoxy-benzyl)-thiazol-2-ylamine

A mixture of 2-chloro-3-(5-fluoro-2-methoxy-phenyl)-propionaldehyde(crude from above) and thiourea (29.2 g, 0.384 mol) in ethanol (400 mL)was heated to reflux for 15 hours. The solvent was removed and theresidue was diluted with dichloromethane (300 mL), sodium hydroxide (10%aqueous solution, 150 mL) and water (200 mL). The aqueous phase wasextracted twice with dichloromethane (150 mL). The combined organiclayer was washed with water, a saturated aqueous solution of sodiumchloride, dried over sodium sulfate, and evaporated to dryness. Thecrude product was recrystallized from a mixture of ethyl acetate andhexanes to give the pure product (6.5 g, 0.027 mol, 6.2% from5-fluoro-2-methoxy-phenylamine). ESI-MS m/z calc. 238.1. found 239.2(M+1)⁺ ¹H NMR (CDCl₃): δ 6.90-6.81 (m, 2H), 6.79-6.76 (m, 2H), 4.75 (br,2H), 3.91 (s, 2H), 3.82 (s, 3H).

a) 2-Methoxy-3-nitro-pyridine

A suspension of sodium methoxide (40.5 g, 0.750 mol) in 200 mL ofmethanol was slowly added to a solution of 2-chloro-3-nitro-pyridine(79.3 g, 0.500 mol) in 800 mL of methanol at 0° C. The reaction mixturewas stirred for 4 hours and then poured into 1000 g of ice. Theresulting precipitate was filtered, washed with water, and dried to give2-methoxy-3-nitro-pyridine (70. g, 0.45 mmol, 90%) as a white solid.

b) 2-Methoxy-pyridin-3-ylamine

A solution of 2-methoxy-3-nitro-pyridine (70. g, 0.45 mol) in methanol(700 mL) containing palladium on carbon (7 g, 10%) was stirred under anatmosphere of hydrogen for 15 hours. The catalyst was filtered andwashed with methanol. The filtrate was evaporated to dryness to givecrude 2-methoxy-pyridin-3-ylamine (48 g, 0.39 mol, 87%), which was useddirectly in the next step.

c) 2-Chloro-3-pyridin-3-yl-propionaldehyde

A solution of sodium nitrite (20. g, 0.28 mol) in water (100 mL) wasslowly added to a solution of 2-methoxy-pyridin-3-ylamine (36 g, 0.25mol) in hydrochloric acid (20% aqueous solution, 60 mL) at 0° C. Afterstirring for 10 minutes, a cooled (0° C.) solution of acrolein (25 mL,0.37 mol) in acetone (25 mL) containing calcium oxide (5 g, 0.09 mol)was slowly added to the reaction mixture. This was then followed by asolution of cuprous chloride (2.5 g, 0.025 mol) in acetone (25 mL)containing hydrochloric acid (20% aqueous solution, 5 mL). The mixturewas stirred at 0 to 30° C. for 3 hours, and then extracted three timeswith dichloromethane (150 mL). The combined organic layers were washedwith a saturated aqueous solution of sodium bicarbonate, a saturatedaqueous solution of sodium chloride, dried over sodium sulfate,filtered, and evaporated to dryness to give a black viscous oil. Thecrude product was passed through a short silica column to give a crudeproduct, which was used directly in the next step.

d) 5-(2-Methoxy-pyridin-3-ylmethyl)-thiazol-2-ylamine

A mixture of 2-chloro-3-pyridin-3-yl-propionaldehyde (crude from above)and thiourea (14.8 g, 0.194 mol) in ethanol (200 mL) was heated toreflux overnight. The solvent was removed and the residue was dilutedwith dichoromethane (1.2 L) and then washed with sodium hydroxide (10%aqueous solution, 400 mL) and water (200 mL). The organic layer wasextracted three times with hydrochloric acid (5% aqueous solution, 400mL) and the combined aqueous layer was brought to between pH 9 and 10with sodium hydroxide (10% aqueous solution). The resulting precipitatewas filtered to give the crude product, which was recrystallized, fromethyl acetate and hexanes to give the pure product (5.1 g, 0.023 mol,5.6% from 2-methoxy-pyridin-3-ylamine). ESI-MS m/z talc. 221.1. found222.2 (M+1)⁺.

a) 2-Bromo-1-(chloro-phenyl)-ethanone

Bromine (3.8 mL, 65 mmol) was added dropwise to a solution of1-(2-chloro-phenyl)-ethanone (10. g, 65 mmol) in acetic acid (75 mL) at0° C. The mixture was then warmed to room temperature and stirredovernight. The mixture was evaporated to dryness and used in the nextstep without further purification.

N′-[5-(2-Chloro-benzoyl)-thiazol-2-yl]-N,N-dimethyl-formamidine. Amixture of thiourea (4.95 g, 65.0 mmol) anddimethoxymethyl-dimethyl-amine (23.2 g, 195 mmol) in methanol (80 mL)was heated under reflux for 30 minutes. After allowing the mixture tocool, triethylamine (19.8 g, 195 mmol) and a solution of2-bromo-1-(chloro-phenyl)-ethanone (crude from last step) in methanol(50 mL) were added. The mixture was heated to reflux for 4 hours. Thesolvent was removed and the residue was used directly in the nextprocedure.

b) (2-Amino-thiazol-5-yl)-(2-chloro-phenyl)-methanone

The crudeN′-[5-(2-chloro-benzoyl)-thiazol-2-yl]-N,N-dimethyl-formamidine wasdissolved in 10% aqueous hydrochloric acid (150 mL) and heated to 70° C.for 4 hours. The precipitate was filtered, washed with ether, and thensuspended in a 10% aqueous sodium carbonate solution (250 mL). Thesuspension was stirred for 1 hour and the precipitate was filtered,washed with ether, and dried in air to give(2-amino-thiazol-5-yl)-(2-chloro-phenyl)-methanone as a brown solid (8.5g, 36 mmol, 55% from 1-(2-chloro-phenyl)-ethanone). ESI-MS m/z calc.238.0. found 239.3 (M+1)⁺ ¹H NMR (DMSO): δ: 7.252 (s, 1H), 7.420-7.553(m, 4H), 8.345 (s, 2H).

a) 2-Chloro-3-(2-chloro-phenyl)-propionaldehyde

To a solution of 2-chloroaniline (12.7 g, 100. mmol) in hydrochloricacid (20% aqueous solution, 40 mL) was added dropwise a solution ofsodium nitrite (7.5 g, 110 mmol) in water (20 mL) at 0 to 5° C. Afterstirring for 10 minutes, a cooled (0° C.) solution of acrolein (15 g,270 mmol) in acetone (100 mL) containing calcium oxide (2.0 g, 36 mmol)was added gradually, and then followed by a solution of cuprous chloride(1 g, 10 mmol) in acetone (10 mL) containing hydrochloric acid (20%aqueous solution, 2 mL). The mixture was stirred at 0 to 30° C. for 3hours and then extracted three times with dichloromethane (100 mL). Thecombined organic layers were washed with a saturated aqueous solution ofsodium bicarbonate followed by a saturated aqueous solution of sodiumchloride. The organic layer was separated, dried over sodium sulfate,and evaporated to dryness to give a black viscous oil. The crude productwas passed through a short silica gel column to give 12 g of crudeproduct, which was used directly in the next step.

b) 5-(2-Chloro-benzyl)-thiazol-2-ylamine

A mixture of 2-chloro-3-(2-chloro-phenyl)-propionaldehyde (12 g, crudefrom above) and urea (6.0 g, 0.10 mol) in ethanol (120 mL) was heated toreflux overnight. The solvent was evaporated to dryness. The residue wasdiluted with dichloromethane (120 mL) and then washed with sodiumhydroxide (10% aqueous solution, 50 mL) and water (30 mL). The organiclayer was extracted three times with hydrochloric acid (5% aqueoussolution, 120 mL). The combined aqueous layer was adjusted with a 10%aqueous solution of sodium hydroxide to between pH 9 and 10 and thenextracted three times with dichloromethane (150 mL). The organic layerswere combined, dried over sodium sulfate, evaporated to dryness, andpurified by silica gel column chromatography to yield a yellow solid.(5.2 g, 0.023 mol, 23% from 2-chloroaniline). ESI-MS m/z calc. 224.0.found 225.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 4.07 (s, 2H), 4.90 (bs, 2H), 6.80(s, 1H), 7.37-7.15 (m, 4H).

6) 5-(2-methoxy-benzyl)-thiazol-2-ylamine

5-(2-methoxy-benzyl)-thiazol-2-ylamine was prepared in a manneranalogous to that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/zcalc. 220.1. found 221.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.26-7.19 (m, 1H), 7.15(d, J=6.8 Hz, 1H), 6.90-6.85 (m, 2H), 6.79 (s, 1H), 4.77 (bs, 2H), 3.93(s, 2H), 3.84 (s, 3H).

7) 5-(3-Chloro-benzyl)-thiazol-2-ylamine

5-(3-Chloro-benzyl)-thiazol-2-ylamine was prepared in a manner analogousto that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/z calc.224.0. found 225.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.26-7.21 (m, 3H), 7.10 (d,J=6.8 Hz, 1H), 6.81 (s, 1H), 4.82 (bs, 2H), 3.93 (s, 2H).

8) 5-(4-Chloro-benzyl)-thiazol-2-ylamine

5-(4-Chloro-benzyl)-thiazol-2-ylamine was prepared in a manner analogousto that of 5-(2-chloro-benzyl)-thiazol-2-ylamine. ESI-MS m/z calc.224.0. found 225.2 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.26 (d, J=8.4 Hz, 2H), 7.14(d, J=8.4 Hz, 2H), 6.79 (s, 1H), 4.85 (bs, 2H), 3.92 (s, 2H).

9) 5-(2-Cyano-benzyl)-thiazol-2-ylamine

5-(2-Cyano-benzyl)-thiazol-2-ylamine was prepared in a manner analogousto that of 5-(2-chloro-benzyl)-thiazol-2-ylamine (12 g, 56 mmol, 11%from 2-cyanoaniline). ESI-MS m/z calc. 215.05. found 216.16 (M+1)⁺ ¹HNMR (CDCl₃): δ 7.64 (d, 1H), 7.54 (t, 1H), 7.34 (m, 2H), 6.87 (s, 1H),4.89 (br, 2H), 4.19 (s, 2H).

a) 2-Chloro-3-(2-methoxy-phenyl)-propionaldehyde

A solution of 2-methoxylaniline (24.6 g, 0.200 mol) in hydrochloric acid(20% aqueous solution, 80 mL) was slowly added to a solution of sodiumnitrite (15 g, 0.22 mol) in water (40 mL) at 0 to 5° C. After stirringfor 10 minutes, a cooled (0° C.) solution of acrolein (30 g, 0.56 mol)in acetone (200 mL) containing calcium oxide (4.0 g, 72 mmol) was slowlyadded, followed by a solution of cuprous chloride (2.0 g, 20 mmol) inacetone (20 mL) containing hydrochloric acid (20% aqueous solution, 4mL). The mixture was stirred at 0 to 30° C. for 3 hours, and thenextracted with three 150 mL portions of dichloromethane. The combinedorganic layers were washed with a saturated aqueous solution of sodiumbicarbonate, a saturated aqueous solution of sodium chloride, dried oversodium sulfate, filtered, and concentrated to give a black viscous oil.The crude product was passed through a short silica column to give 10 gof crude product, which was used directly in the next procedure.

b) 5-(2-methoxy-benzyl)-oxazol-2-ylamine

A mixture of 2-chloro-3-(2-methoxylphenyl)-propionaldehyde (10 g, crudefrom above) and urea (9.6 g, 0.16 mol) was dissolved in ethanol (250 mL)and then heated to reflux overnight. The solvent was evaporated todryness. The residue was diluted with dichloromethane (250 mL) and thenwashed with sodium hydroxide (10% aqueous solution, 100 mL) and water(50 mL). The organic layer was extracted three times with hydrochloricacid (5% aqueous solution, 250 mL). The combined aqueous layers wereadjusted to pH 9 to 10 with a 10% aqueous solution of sodium hydroxideand then extracted three times with dichloromethane (300 mL). Theorganic layer was separated, dried over sodium sulfate, and evaporatedto dryness. The crude product was purified by silica gel columnchromatography to yield the yellow-red solid product. (0.72 g, 0.35%from 2-methoxyaniline). ESI-MS m/z calc. 204.1. found 205.1 (M+1)⁺ ¹HNMR (CDCl₃) δ 7.26-7.20 (m, 1H), 7.14 (d, J=7.2 Hz, 1H), 6.91-6.86 (m,2H), 6.35 (s, 1H), 4.49 (bs, 2H), 3.85 (s, 2H), 3.82 (s, 3H).

11)

5-(2-Chloro-benzyl)-oxazol-2-ylamine

5-(2-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid. (3.5 g, 8.4% from 2-chloroaniline).ESI-MS m/z calc. 208.0. found 209.1 (M+1)⁺ ¹H NMR (CDCl₃) δ 7.37-7.18(m, 4H), 6.40 (s, 1H), 4.66 (bs, 2H), 3.97 (s, 2H).

12)

5-(3-Chloro-benzyl)-oxazol-2-ylamine

5-(3-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid (1.2 g, 2.9% from 3-chloroaniline).ESI-MS m/z calc. 208.0. found 209.2 ¹H NMR (CDCl₃) δ 7.26-7.22 (m, 3H),7.10 (d, J=6.0 Hz, 1H), 6.44 (s, 1H), 4.73 (bs, 2H), 3.82 (s, 2H).

13)

5-(4-Chloro-benzyl)-oxazol-2-ylamine

5-(4-Chloro-benzyl)-oxazol-2-ylamine was prepared in a manner analogousto that of the preparation of 5-(2-methoxy-benzyl)-oxazol-2-ylamine toyield the product as a yellow solid (1.6 g, yield 3.86% from4-chloroaniline). ESI-MS m/z calc. 208.0. found 209.1 ¹H NMR (CDCl₃) δ7.27 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.0 Hz, 2H), 6.38 (s, 1H), 4.66 (bs,2H), 3.81 (s, 2H).

a) 2-Bromo-3-phenylpropionaldehyde

A solution of bromine (15.2 g, 95.1 mmol) in 30 mL of dichloromethanewas added to a solution of 3-phenyl-propionaldehyde (13.4 g, 100 mmol)in dichloromethane (150 mL) at 0° C. over 20 minutes. The reactionmixture was allowed to stir for 2 hours and then a saturated aqueoussolution of sodium bicarbonate (100 mL) was added to the mixture. Theorganic layer was separated and the aqueous layer was washed withdichloromethane (50 mL). The combined organic layers were washed withwater, a saturated aqueous solution of sodium chloride, and thenevaporated to dryness to give an orange oil (14.2 g), which was useddirectly in the next step.

b) 5-Benzyl-oxazol-2-ylamine

A mixture of 2-bromo-3-phenylpropionaldehyde (14.2 g, crude from above)and urea (7.2 g, 0.12 mol) were heated to reflux for 15 hours in 200 mLof ethanol. The solvent was evaporated to dryness and the residue wasdiluted with dichloromethane (250 mL) and then washed with sodiumhydroxide (10% aqueous solution, 100 mL) and water (50 mL). The organiclayer was extracted three times with hydrochloric acid (5% aqueoussolution, 250 mL). The combined aqueous layers were adjusted to betweenpH 9 to 10 with a 10% aqueous solution of sodium hydroxide and thenextracted three times with dichloromethane (300 mL). The organic layerwas dried over sodium sulfate, evaporated to dryness, and purified bysilica gel column chromatography to give a pale yellow solid. (1.6 g,9.2 mmol, 9.2% from 3-phenyl-propionaldehyde). ESI-MS m/z calc. 174.1.found 175.1 ¹H NMR (CDCl₃) δ 7.32-7.22 (m, 5H), 6.39 (s, 1H), 4.72 (bs,2H), 3.84 (s, 2H).

a) 2-Bromo-3-phenylpropionaldehyde

A solution of bromine (15.2 g, 95.1 mmol) in 30 mL of dichloromethanewas added to a solution of 3-phenyl-propionaldehyde (13.4 g, 100 mmol)in dichloromethane (150 mL) at 0° C. over 20 minutes. The reactionmixture was allowed to stir for 2 hours and then a saturated aqueoussolution of sodium bicarbonate (100 mL) was added to the mixture. Theorganic layer was separated and the aqueous layer was washed withdichloromethane (50 mL). The combined organic layers were washed withwater, a saturated aqueous solution of sodium chloride, and thenevaporated to dryness to give an orange oil (14.2 g), which was useddirectly in the next step.

b) 5-benzyl-thiazol-2-ylamine

A mixture of 2-bromo-3-phenylpropionaldehyde (14.2 g, crude from above)and urea (7.2 g, 0.12 mol) were heated to reflux for 15 hours in 200 mLof ethanol. The solvent was evaporated to dryness and the residue wasdiluted with dichloromethane (250 mL) and then washed with sodiumhydroxide (10% aqueous solution, 100 mL) and water (50 mL). The organiclayer was extracted three times with hydrochloric acid (5% aqueoussolution, 250 mL). The combined aqueous layers were brought to pH 9 to10 with a 10% aqueous solution of sodium hydroxide and then extractedthree times with dichloromethane (300 mL). The organic layer was driedover sodium sulfate, evaporated to dryness, and purified by silica gelcolumn chromatography to give a pale yellow solid. (5.2 g, 27 mmol, 27%from 3-phenyl-propionaldehyde). ESI-MS m/z calc. 190.1. found 191.2 ¹HNMR (CDCl₃) δ 7.32-7.21 (m, 5H), 6.79 (s, 1H), 4.91 (bs, 2H), 3.95 (s,2H).

a) 2-Chloro-3-(2-chloro-phenyl)-propionaldehyde

A solution of sodium nitrite (15 g, 0.22 mol) in water (40 mL) wasslowly added to a solution of 2-chloroaniline (25.5 g, 0.200 mol) inhydrochloric acid (20% aqueous solution, 100 mL) at 0 to 5° C. Themixture was stirred for ten minutes and then poured into a cooled (0°C.) solution of acrolein (30. g, 0.56 mol) in acetone (200 mL)containing calcium oxide (4.0 g, 72 mmol), followed by a solution ofcuprous chloride (2.0 g, 20 mmol) in acetone (20 mL) containinghydrochloric acid (20% aqueous solution, 4 mL). The mixture was stirredfor 3 hours at room temperature, and then extracted three times withdichloromethane (150 mL). The combined organic layers were washed with asolution of saturated aqueous sodium chloride, dried over sodiumsulfate, filtered, and evaporated to dryness to give a black viscous oilthat was used directly in the next procedure.

b) [2-(2-Chloro-phenyl)-1-methyliminomethyl-ethyl]-methyl-amine

A solution of methylamine in methanol (27%, 69 g) was slowly added to asolution of 2-chloro-3-(2-chloro-phenyl)-propionaldehyde indichloromethane (20 mL). The reaction mixture was allowed to stir for 12hours and then used immediately in the next procedure.

c) 5-(2-Chloro-benzyl)-1-methyl-1H-imidazol-2-ylamine

A solution of cyanamide in water (50%, 150 mL) was added to a boilingsolution of [2-(2-chloro-phenyl)-1-methyliminomethyl-ethyl]-methyl-aminein methanol and dichloromethane. The pH was brought to 4.5 by thecontinual addition of an aqueous solution of sulfuric acid (9 M). Themixture was refluxed for 2 hours, allowed to cool to room temperature,and adjusted to pH 9 through the addition of powdered sodiumbicarbonate. The mixture was extracted three times with dichloromethane(200 mL) and the combined organic layers were extracted three times withhydrochloric acid (20% aqueous solution, 150 mL). The aqueous solutionwas adjusted to pH 10 with sodium hydroxide (10% aqueous solution) andextracted three times with dichloromethane (150 mL). The combinedorganic layers were washed with a saturated aqueous solution of sodiumchloride, dried over sodium sulfate, filtered, and evaporated to drynessto give a black solid which was purified by column chromatography toyield the product (5.0 g, 0.23 mmol, 11% from 2-chloroaniline) as abrown solid. ESI-MS m/z calc. 221.1. found 222.3 (M+1)⁺ ¹H NMR (CDCl₃):δ 7.30-7.37 (m, 1H), 7.15-7.18 (m, 2H), 7.03-7.06 (m, 1H), 6.43 (s, 1H),3.94 (s, 2H), 3.80 (br, 2H), 3.15 (s, 3H).

a) 2-Bromo-3-oxo-hexanoic acid ethyl ester

3-Oxo-hexanoic acid ethyl ester (4.0 mL, 25 mmol) and magnesiumperchlorate (1.7 g, 7.6 mmol) were placed in 500 mL of ethyl acetate andallowed to stir for 5 minutes. N-Bromosuccinimide (4.7 g, 26 mmol) wasadded and the reaction mixture was allowed to stir for 15 minutes, atwhich time thin-layer chromatography (10% ethyl acetate in hexanes,silica gel, 254 nm irradiation) indicated the reaction was complete. Thereaction mixture was diluted with 500 mL of ethyl ether and washed threetimes with an equal volume of saturated aqueous sodium chloride. Theorganic layer was then dried over sodium sulfate and evaporated todryness. This material was used in the next step without furtherpurification.

b) Amino-4-propyl-thiazole-5-carboxylic acid ethyl ester

2-Bromo-3-oxo-hexanoic acid ethyl ester (5.9 g, 25 mmol), was dissolvedin 60 mL of ethanol containing triethylamine (4.2 mL, 30 mmol) andthiourea (1.9 g, 25 mmol). The colorless solution was protected fromlight and allowed to stir for 16 hours. The resulting red suspension wasevaporated to dryness and dissolved in a minimum of dichloromethane.This solution was washed three times with an equal volume of a saturatedaqueous solution of sodium bicarbonate, followed by a saturated aqueoussolution of sodium chloride. The organic layer was separated andfiltered to remove a fine red precipitate which remained suspended inthe organic phase. The solvent was removed and then the solid wasdissolved in a minimum of 50/50 (v/v) ethyl acetate and 1 N aqueoussolution of hydrochloric acid. The layers were separated and the aqueouslayer was washed with an equal volume of ethyl acetate. After discardingthe organic layers, the aqueous layer was then placed in an ice bathwith an equal volume of ethyl acetate. Sodium hydroxide (1N) was thenslowly added with vigorous swirling until the aqueous phase was basic.The layers were separated and the aqueous layer was washed twoadditional times with ethyl acetate. The combined organic layers werewashed three times with an equal volume of a solution of saturatedaqueous sodium bicarbonate followed by a solution of saturated aqueoussodium chloride. The organic layer was then dried over sodium sulfateand evaporated to dryness to yield a pale yellow solid (1.8 g, 8.4 mmol,34%) ESI-MS m/z calc. 214.1. found 215.3 (M+1)⁺ Retention time 1.90minutes.

2-Bromo-1-(2-methoxy-phenyl)-ethanone (0.6388 g, 2.789 mmol) andthiourea (0.2289 g, 3.007 mmol) were dissolved in a 20 mL of ethanol.The reaction mixture was allowed to stir overnight at room temperature.The ethanol was evaporated to dryness and the crude product wasdissolved in a minimum of dichloromethane. The crude product was thenextracted twice with 1M sodium hydroxide and once with a saturatedaqueous solution of sodium chloride. The organic layer was then driedover sodium sulfate, filtered, and evaporated to dryness to yield thepure product (0.529 g, 2.56 mmol, 92.0%). ESI-MS m/z calc. 206.3. found207.1 (M+1)⁺ Retention time 1.86 minutes. ¹H NMR (400 MHz, CD₃CN) δ 3.91(s, 3H), 5.54 (s, 2H), 6.97-7.02 (m, 1H), 7.03-7.06 (m, 1H), 7.23 (s,1H), 7.24-7.29 (m, 1H), 8.06 (dd, J=7.8, 1.8 Hz, 1H).

2-Bromo-1-(3-methoxy-phenyl)-ethanone (0.7291 g, 3.183 mmol) andthiourea (0.2665 g, 3.501 mmol) were dissolved in a 20 mL of ethanol.The reaction mixture was allowed to stir overnight at room temperature.The ethanol was evaporated to dryness and the crude product wasdissolved in a minimum of dichloromethane. The crude product was thenextracted twice with 1M sodium hydroxide and once with a saturatedaqueous solution of sodium chloride. The organic layer was then driedover sodium sulfate, filtered, and evaporated to dryness to yield theproduct (0.619 g, 3.00 mmol, 94.3%). ESI-MS m/z calc. 206.3. found 207.0(M+1)⁺ Retention time 1.86 minutes. ¹H NMR (400 MHz, CD₃CN) δ 3.84 (s,3H), 5.67 (s, 2H), 6.85-6.91 (m, 2H), 7.31 (t, J=7.9 Hz, 1H), 7.36-7.43(m, 2H).

2-Bromo-1-phenyl-ethanone (19.9 g, 0.100 mol) and thiourea (7.9 g, 0.10mol) were mixed in a 150 mL of methanol. The reaction mixture was warmedto dissolve the reagents and then allowed to stir overnight at roomtemperature. The methanol was evaporated to dryness and the crudeproduct was dissolved in a minimum of ethyl acetate. The crude productwas then extracted twice with 1M sodium hydroxide and once with asaturated aqueous solution of sodium chloride. The organic layer wasthen dried over sodium sulfate, filtered, and evaporated to dryness toyield the pure product (16.8 g, 0.0953 mol, 95.3%) of product. ESI-MSm/z calc. 176.0. found 177.2 (M+1)⁺ Retention time 1.41 minutes. ¹H NMR(400 MHz, CD₃CN) δ 5.73 (s, 2H), 6.87 (s, 1H), 7.28-7.34 (m, 1H),7.36-7.43 (m, 2H), 7.79-7.85 (m, 2H).

a) 2-Pyridin-3-yl-butyric acid ethyl ester

Pyridin-3-yl-acetic acid ethyl ester (22.5 g, 0.136 mol) intetrahydrofuran (50 mL) was slowly added to a suspension of sodiumhydride (60% in mineral oil, 6 g, 0.4 mol) in tetrahydrofuran (50 mL) at0° C. A solution of ethyl iodide (23.4 g, 0.150 mol) in tetrahydrofuran(50 mL) was added to and the reaction mixture was allowed to stirovernight at room temperature. The mixture was poured into ice and theaqueous layer was extracted three times with ethyl acetate. The organiclayer washed with a saturated aqueous solution of sodium chloride, driedover sodium sulfate, and evaporated to dryness. The crude residue waspurified by preparative reverse phase liquid chromatography (13.5 g,0.0699 mol, 51.3%). ESI-MS m/z calc. 193.1. found 194.2 (M+1)⁺ ¹H NMR(CDCl₃) δ: 9.42-9.39 (m, 2H), 8.58-8.55 (m, 1H), 8.17-8.13 (m, 1H),5.09-4.97 (m, 2H), 4.35 (t, 1H, J=7.6 Hz), 3.04-2.95 (m, 1H), 2.74-2.65(m, 1H), 2.10 (t, 3H, J=7.2 Hz), 1.79 (t, 3H, J=7.6 Hz).

b) 2-Pyridin-3-yl-butyric acid

A mixture of 2-Pyridin-3-yl-butyric acid ethyl ester (8 g, 0.04 mol) anda 20% aqueous solution of hydrochloric acid (50 mL) was heated to refluxfor 3 hours. The solvent evaporated to dryness to yield the desiredproduct (5 g, 0.02 mol, 50%), ESI-MS m/z calc. 165.1. found 166.5 (M+1)⁺¹H NMR (DMSO-d₆): δ 8.86 (s, 1H), 8.80 (d, 1H, J=5.6 Hz), 8.47 (d, 1H,J=8.0 Hz), 7.97 (dd, 1H, J=5.6, 8.0 Hz), 3.83 (t, 1H, J=8.0 Hz),2.07-2.04 (m, 1H), 1.82-1.80 (m, 1H), 0.80 (t, 3H, J=7.6 Hz).

N-[5-(2-Chloro-benzoyl)-thiazol-2-yl]-2-phenyl-butyramide (102 mg, 0.265mmol) was suspended in 1 mL of anhydrous methanol. Sodium borohydride(30.3 mg, 0.801 mmol) was slowly added and the resulting pale yellowsolution was allowed to stir for 1 hour at room temperature. Afterstirring for one hour a second aliquot of sodium borohydride (30.3 mg,0.801 mmol) was added. The reaction mixture was allowed to stir for anadditional hour and then the crude product was evaporated to dryness andthen dissolved in a minimum of ethyl acetate. The organic layer waswashed three times with an equal volume of 1N hydrochloric acid,saturated aqueous sodium bicarbonate, and saturated aqueous sodiumchloride. The organic layer was then dried over sodium sulfate,filtered, and evaporated to dryness. The crude product was furtherpurified by reverse-phase preparative liquid chromatography to yield thepure product (46 mg, 0.12 mmol, 45%) ESI-MS m/z calc. 386.1. found 387.3(M+1)⁺ Retention time 3.83 minutes.

5-(2-Chloro-benzyl)-thiazol-2-ylamine (45 mg, 0.20 mmol) anddiphenyl-acetic acid (42 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (1 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (10.3 mg, 0.0246 mmol,12%). ESI-MS m/z calc. 418.1. found 419.2 (M+1)⁺ Retention time 3.85minutes. ¹H NMR (400 MHz, DMSO-d₆) δ 4.19 (s, 2H), 5.30 (s, 1H),7.23-7.47 (m, 15H).

2-Amino-5,6-dihydro-4H-benzothiazol-7-one (34 mg, 0.20 mmol) and2-phenyl-butyric acid (33 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (1 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (31 mg, 0.099 mmol,49%). ESI-MS m/z calc. 314.1. found 315.3 (M+1)⁺ Retention time 2.90minutes.

(2-Amino-thiazol-4-yl)-acetic acid methyl ester (45 mg, 0.20 mmol) anddiphenyl-acetic acid (37 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (2 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (29.4 mg, 0.0802 mmol,40%). ESI-MS m/z calc. 380.1. found 381.3 (M+1)⁺ Retention time 3.28minutes.

2-Amino-thiazole-4-carboxylic acid methyl ester (32 mg, 0.20 mmol) anddiphenyl-acetic acid (42 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (1 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (19 mg, 0.052 mmol,26%). ESI-MS m/z calc. 366.1. found 367.1 (M+1)⁺ Retention time 3.34minutes.

5-(2-Methoxy-pyridin-3-ylmethyl)-thiazol-2-ylamine (44 mg, 0.20 mmol)and 2-phenyl-butyric acid (33 mg, 0.20 mmol) were dissolved inacetonitrile (1 mL) containing triethylamine (84.1 μL, 0.600 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (15 mg, 0.041 mmol,21%). ESI-MS m/z calc. 367.1. found 368.1 (M+1)⁺ Retention time 3.24minutes.

5-(2-Chloro-benzyl)-thiazol-2-ylamine (450 mg, 2.0 mmol) andbromo-phenyl-acetic acid (430 mg, 2.0 mmol) were dissolved inacetonitrile (20 mL) containing triethylamine (280 μL, 2.0 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (836 mg, 2.2 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified by silicagel chromatography using a gradient of 10-30% ethyl acetate in hexanesto yield a pale yellow solid (633 mg, 1.50 mmol, 75.0%). ESI-MS m/zcalc. 420.0. found 421.2 (M+1)⁺ Retention time 3.63 minutes.

5-(2-Chloro-benzyl)-thiazol-2-ylamine (0.484 g, 2.16 mmol) and2-bromo-butyric acid (0.360 g, 2.16 mmol) were dissolved in acetonitrile(20 mL) containing triethylamine (302 μL, 2.16 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (1.15 g, 3.02 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified by silicagel chromatography. ESI-MS m/z calc. 372.0. found 373.2 (M+1)⁺ Retentiontime 3.41 minutes.

5-(4-Chloro-benzyl)-oxazol-2-ylamine (42 mg, 0.20 mmol) andcyclohexyl-phenyl-acetic acid (44 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (1 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (9.6 mg, 0.023 mmol,12%). ESI-MS m/z calc. 408.9. found 409.4 (M+1)⁺ Retention time 3.76minutes.

5-(2-Chloro-benzyl)-1-methyl-1H-imidazol-2-ylamine (44 mg, 0.20 mmol)and 2-phenyl-butyric acid (33 mg, 0.20 mmol) were dissolved inN,N-dimethylformamide (1 mL) containing triethylamine (84.1 μL, 0.600mmol). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (13 mg, 0.035 mmol,18%). ESI-MS m/z calc. 367.2. found 368.1 (M+1)⁺ Retention time 2.42minutes.

6-Ethoxy-benzothiazol-2-ylamine (39 mg, 0.20 mmol) and 2-phenyl-butyricacid (33 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (1 mL)containing triethylamine (84.1 μL, 0.600 mmol).O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (84 mg, 0.22 mmol) was added and the solution wasallowed to stir for 16 hours. The crude product was purified byreverse-phase preparative liquid chromatography (17 mg, 0.050 mmol,25%). ESI-MS m/z calc. 340.1. found 340.9 (M+1)⁺ Retention time 3.55minutes.

4-Phenyl-thiazol-2-ylamine (35.2 mg, 0.200 mmol) and 4-methyl-benzoylchloride (30.9 mg, 0.200 mmol) were dissolved in 1 mL of pyridine. Thereaction mixture was stirred at room temperature overnight and thenpurified by reverse-phase preparative liquid chromatography (20.6 mg,0.0635 mmol, 31.8%). ESI-MS m/z calc. 294.1. found 295.2 (M+1)⁺Retention time 3.55 minutes.

5-Phenyl-thiazol-2-ylamine (35.2 mg, 0.200 mmol) and 4-methyl-benzoylchloride (30.9 mg, 0.200 mmol) were dissolved in 1 mL of pyridine. Thereaction mixture was stirred at room temperature overnight and thenpurified by reverse-phase preparative liquid chromatography (10.4 mg,0.0353 mmol, 17.7%). ESI-MS m/z calc. 294.1. found 295.4 (M+1)⁺Retention time 3.40 minutes.

4-(2-Methoxy-phenyl)-thiazol-2-ylamine (41.3 mg, 0.200 mmol) and4-methyl-benzoyl chloride (30.9 mg, 0.200 mmol) were dissolved in 1 mLof pyridine. The reaction mixture was stirred at room temperatureovernight and then purified by reverse-phase preparative liquidchromatography (7.32 mg, 0.0226 mmol, 11.3%). ESI-MS m/z calc. 324.1.found 325.2 (M+1)⁺ Retention time 3.75 minutes.

4-(2-Methoxy-phenyl)-thiazol-2-ylamine (41.3 mg, 0.200 mmol) and benzoylchloride (28.1 mg, 0.200 mmol) were dissolved in 1 mL of pyridine. Thereaction mixture was stirred at room temperature overnight and thenpurified by reverse-phase preparative liquid chromatography (11.4 mg,0.0367 mmol, 18.4%). ESI-MS m/z calc. 310.1. found 311.2 (M+1)⁺Retention time 3.55 minutes.

4,5-Diphenyl-thiazol-2-ylamine (50.5 mg, 0.200 mmol) and benzoylchloride (28.1 mg, 0.200 mmol) were dissolved in 1,4-dioxane (2 mL)containing triethylamine (84.1 μL, 0.600 mmol). The reaction mixture wassubjected to microwave irradiation for 5 minutes at 200° C. The crudeproduct was filtered, evaporated to dryness, dissolved in 1 mL ofdimethylsulfoxide and purified by reverse-phase preparative liquidchromatography (7.21 mg, 0.0202 mmol, 10.1%). ESI-MS m/z calc. 356.1.found 357.2 (M+1)⁺ Retention time 3.95 minutes.

4,5-Diphenyl-thiazol-2-ylamine (50.5 mg, 0.200 mmol) and4-methyl-benzoyl chloride (30.9 mg, 0.200 mmol) were dissolved in1,4-dioxane (2 mL) containing triethylamine (84.1 μL, 0.600 mmol). Thereaction mixture was subjected to microwave irradiation for 5 minutes at200° C. The crude product was filtered, evaporated to dryness, dissolvedin 1 mL of dimethylsulfoxide and purified by reverse-phase preparativeliquid chromatography (18.6 mg, 0.0502 mmol, 25.1%). ESI-MS m/z calc.370.1. found 371.2 (M+1)⁺ Retention time 4.13 minutes.

4,5-Diphenyl-thiazol-2-ylamine (50.5 mg, 0.200 mmol) and4-methoxy-benzoyl chloride (34.1 mg, 0.200 mmol) were dissolved in1,4-dioxane (2 mL) containing triethylamine (84.1 μL, 0.600 mmol). Thereaction mixture was stirred overnight at room temperature. The crudeproduct was filtered, evaporated to dryness, dissolved in 1 mL ofdimethylsulfoxide and purified by reverse-phase preparative liquidchromatography (12.5 mg, 0.0323 mmol, 16.2%). ESI-MS m/z calc. 386.1.found 387.2 (M+1)⁺ Retention time 3.95 minutes.

4-p-Tolyl-thiazol-2-ylamine (38.1 mg, 0.200 mmol) and 4-methyl-benzoylchloride (30.9 mg, 0.200 mmol) were dissolved in 1,4-dioxane (2 mL)containing triethylamine (84.1 μL, 0.600 mmol). The reaction mixture wasstirred overnight at room temperature. The crude product was filtered,evaporated to dryness, dissolved in 1 mL of dimethylsulfoxide andpurified by reverse-phase preparative liquid chromatography (13.5 mg,0.0438, 21.9%). ESI-MS m/z calc. 308.1. found 309.0 (M+1)⁺ Retentiontime 3.72 minutes.

5-Methyl-thiazol-2-ylamine (22.8 mg, 0.200 mmol) and 4-methyl-benzoylchloride (30.9 mg, 0.200 mmol) were dissolved in 1,4-dioxane (2 mL)containing triethylamine (84.1 μL, 0.600 mmol). The reaction mixture wasstirred overnight at room temperature. The crude product was filtered,evaporated to dryness, dissolved in 1 mL of dimethylsulfoxide andpurified by reverse-phase preparative liquid chromatography (9.29 mg,0.0400, 20.0%). ESI-MS m/z calc. 232.1. found 233.2 (M+1)⁺ Retentiontime 2.65 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-2-phenyl-acetamide (42 mg,0.10 mmol) was dissolved in 5 mL of methanol. The reaction vessel wassealed and then subjected to microwave irradiation for 15 minutes at125° C. The crude mixture was evaporated to dryness, dissolved in 1 mLof dimethylsulfoxide and purified by reverse-phase preparative liquidchromatography (16 mg, 0.043, 43%). ESI-MS m/z calc. 372.1. found 373.2(M+1)⁺ Retention time 3.43 minutes. ¹H NMR (400 MHz, CD₃CN) δ 3.42 (s,3H), 4.23 (s, 2H), 4.94 (s, 1H), 7.21-7.53 (m, 10H)

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-2-phenyl-acetamide (42 mg,0.10 mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containing1.0 mL of methylamine (2.0 M in tetrahydrofuran, 2.0 mmol). The reactionvessel was sealed and then subjected to microwave irradiation for 5minutes at 80° C. The crude mixture was evaporated to dryness, dissolvedin 1 mL of dimethylsulfoxide and purified by reverse-phase preparativeliquid chromatography (29 mg, 0.078, 78%). ESI-MS m/z calc. 371.1. found372.2 (M+1)⁺ Retention time 2.33 minutes. ¹H NMR (400 MHz, MeOD) δ 2.66(s, 3H), 4.23 (s, 2H), 5.09 (s, 1H), 7.13-7.56 (m, 10H).

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-2-phenyl-acetamide (42 mg,0.10 mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containingmorpholine (174 mg, 2.00 mmol). The reaction vessel was sealed and thensubjected to microwave irradiation for 5 minutes at 80° C. The crudemixture was evaporated to dryness and purified by reverse-phasepreparative liquid chromatography (31 mg, 0.072, 72%). ESI-MS m/z calc.427.1. found 428.0 (M+1)⁺ Retention time 2.40 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-2-phenyl-acetamide (42 mg,0.10 mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containingdimethylamine (90.2 mg, 2.00 mmol). The reaction vessel was sealed andthen subjected to microwave irradiation for 5 minutes at 80° C. Thecrude mixture was evaporated to dryness and purified by reverse-phasepreparative liquid chromatography (13 mg, 0.034, 34%). ESI-MS m/z calc.385.1. found 386.0 (M+1)⁺ Retention time 2.40 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-butyramide (37 mg, 0.10mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containinganiline (186 mg, 2.00 mmol). The reaction vessel was sealed and thensubjected to microwave irradiation for 5 minutes at 80° C. The crudemixture was evaporated to dryness and purified by reverse-phasepreparative liquid chromatography (24 mg, 0.062, 62%). ESI-MS m/z calc.385.1. found 386.1 (M+1)⁺ Retention time 3.48 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-butyramide (37 mg, 0.10mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containingethylamine (2 M in tetrahydrofuran, 1.00 mL, 2.00 mmol). The reactionvessel was sealed and then subjected to microwave irradiation for 5minutes at 80° C. The crude mixture was evaporated to dryness andpurified by reverse-phase preparative liquid chromatography (19 mg,0.056, 56%). ESI-MS m/z calc. 337.1. found 338.0 (M+1)⁺ Retention time2.08 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-butyramide (37 mg, 0.10mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containingpiperidine (170 mg, 2.0 mmol). The reaction vessel was sealed and thensubjected to microwave irradiation for 5 minutes at 80° C. The crudemixture was evaporated to dryness and purified by reverse-phasepreparative liquid chromatography (31 mg, 0.082, 82%). ESI-MS m/z calc.377.1. found 378.2 (M+1)⁺ Retention time 2.21 minutes.

2-Bromo-N-[5-(2-chloro-benzyl)-thiazol-2-yl]-butyramide (37 mg, 0.10mmol) was dissolved in 0.5 mL of N,N-dimethylformamide containingdiethylamine (146 mg, 2.00 mmol). The reaction vessel was sealed andthen subjected to microwave irradiation for 5 minutes at 80° C. Thecrude mixture was evaporated to dryness and purified by reverse-phasepreparative liquid chromatography (14 mg, 0.038, 38%). ESI-MS m/z calc.365.1. found 366.2 (M+1)⁺ Retention time 2.18 minutes.

Analytical data for exemplary compounds of the present invention isrecited below in Table 2 below.

TABLE 2 Cmpd # LC/MS LC/RT 2 309.00 3.00 4 325.00 3.63 5 325.20 3.53 6341.00 3.42 7 341.00 3.47 18 281.20 3.20 21 280.80 3.35 23 329.00 3.3824 310.90 3.58 25 385.20 3.67 28 329.00 3.37 29 335.00 3.50 31 343.203.33 32 309.00 3.13 34 374.00 1.83 35 338.00 1.27 37 419.20 3.92 38419.20 3.85 39 385.30 4.24 47 309.00 3.72 48 295.20 2.83 49 311.00 3.4150 309.20 2.01 51 387.40 2.96 52 371.20 4.13 53 315.00 3.63 55 287.003.53 58 324.20 2.05 66 247.00 2.73 67 295.20 3.52 68 357.20 3.95 69311.00 3.43 70 371.20 4.11 71 371.20 3.94 72 387.20 3.95 73 425.20 4.4174 391.20 4.18 75 295.00 3.45 76 311.00 3.52 77 314.80 3.38 78 311.203.37 79 341.00 3.53 80 340.80 3.49 81 311.20 3.35 82 341.00 3.42 83325.00 3.53 84 340.80 3.38 85 247.00 3.03 86 261.00 3.27 87 261.00 3.3088 295.00 3.55 89 399.40 3.63 90 433.40 3.79 91 425.20 4.24 92 375.403.57 93 383.20 3.57 94 383.40 3.42 95 487.00 4.27 96 337.20 3.53 97371.00 3.72 98 365.00 3.85 99 399.40 4.02 100 349.20 3.28 101 377.403.89 102 411.40 4.07 103 385.20 3.85 104 391.20 2.70 105 387.20 2.78 106391.20 3.03 107 371.20 2.55 108 323.20 2.30 109 323.20 2.33 110 337.202.58 111 321.20 1.98 112 337.00 3.27 113 349.20 3.33 114 363.20 3.57 115351.00 2.88 116 351.00 2.51 117 425.20 4.25 118 489.00 5.32 119 357.203.53 120 371.20 3.72 121 399.40 4.02 122 411.40 4.09 121 385.20 3.87 124419.20 3.84 125 487.20 4.27 126 355.20 3.43 127 357.20 3.52 128 371.003.70 129 399.20 4.00 130 411.20 4.09 131 433.40 3.75 132 421.20 4.05 133413.40 3.57 134 483.20 4.10 135 353.00 3.32 136 367.20 3.50 137 395.203.84 138 381.20 3.67 139 391.20 4.09 140 429.40 3.62 141 279.00 2 66 142409.40 3.76 143 403.40 3 42 144 417.20 3.58 145 339.00 3.02 146 355.003.22 147 383.40 3.52 148 395.00 3.57 149 327.40 2.91 150 313.20 2.91 151409.40 3.72 152 339.00 3.00 153 341.00 3.03 154 355.00 3.20 155 383.203.52 156 395.00 3.57 157 327.40 2.90 158 313.00 2.90 159 315.00 2.81 160315.00 2 96 161 306.20 2.48 162 341.00 2.65 163 350.20 2.53 164 325.207.51 165 391.20 3.68 166 309.20 2.80 167 323.20 3.05 168 337.20 3.22 169363.20 3.53 170 351.20 3.43 171 339.00 7.68 172 385.20 3.45 173 399.203.70 174 413.40 3.87 175 439.40 4.15 176 427.40 4.05 177 453.40 4.30 178415.20 3.40 179 405 40 3.55 180 399.20 3.27 181 335.40 7.83 182 337.007.86 183 351.20 3.03 184 379.40 3.37 185 309.20 2.73 186 409.40 3.70 187471.20 3.85 188 339.20 2.96 189 341.20 3.02 190 355.20 3.18 191 383.203.50 192 395.00 3.55 193 369 20 3.32 194 327.40 2.86 195 313.20 7.85 196563.40 2.65 197 567.20 3.03 198 547.40 2.83 199 537.40 2.51 200 396.203.38 201 — 3.83 202 — 99.00 204 371.00 3.67 205 — 99.00 206 309.20 3.25207 293.00 2.96 208 281.20 2.86 209 321.20 3.35 210 295.20 3.07 211386.00 2.24 212 414.20 2.31 213 386.20 2.24 214 428.00 2.40 215 426.002.33 216 373.20 3.48 217 372.20 2.33 218 374.40 2.03 219 325.20 3.15 770338.00 2.08 221 366.20 2.18 222 338.00 2.03 223 380.00 2.11 224 396.002.33 225 378.20 7.71 226 295.40 3.40 227 345.00 3.62 228 129.00 3.70 229349.00 3.79 230 329.00 3.65 231 325.20 3.48 232 344.80 3.60 233 315.002.91 234 387.20 3.64 235 409.40 3.44 236 383.20 5.11 237 366.70 3.48 238370.90 3.67 239 400.30 2.46 240 454.30 2.70 241 412.30 2.48 242 426.102.55 243 440.10 2.61 244 456.30 2.41 245 498.30 2.76 246 440.10 2.63 247498.10 2.70 248 442.30 2.45 249 505.30 2.78 250 426.30 2.63 251 386.103.48 252 387.10 3.62 253 498.30 3.66 254 462.00 3.02 255 371.90 3.48 256416.30 4.54 257 348.10 4.14 258 362.10 4.22 259 390.30 4.42 260 402.504.44 261 376.30 4.32 262 385.00 3.80 263 387.00 3.83 264 375.10 3.07 265340.90 3.55 266 327.30 3.38 267 325.10 3.70 268 311.10 3.67 269 415.303.38 270 401.30 3.10 271 371.10 3.00 272 400.10 1.62 273 397.30 2.93 274367.10 2.85 275 396.10 1.41 276 362.10 2.56 277 391.10 0.71 278 368.102.42 279 309.10 3.14 280 367.10 3.34 281 381.30 3.28 282 416.50 3.41 283368.10 3.24 284 367.00 3.51 285 385.00 3.60 286 367.00 3.49 287 409.303.93 288 361.10 3.84 289 315.30 2.90

EXAMPLE 6 Assays for Detecting and Measuring ΔF508-CFTR Correction AndPotentiator Properties of Compounds

A) Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 mM forskolin and theCFTR potentiator, genistein (20 mM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 □M forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH. Chloride-free bath Chloride salts in Bath solution:Solution #1 are substituted with gluconate salts. CC2-DMPE: Prepared asa 10 mM stock solution in DMSO and stored at −20° C. DiSBAC₂(3):Prepared as a 10 mM stock in DMSO and stored at −20° C.Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs. for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

B) Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds

1. Ussing Chamber Assay

Ussing chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the £F508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, IA, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(sc)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 mM) and the PDE inhibitor, IBMX (100 mM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 mM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 mM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(sc) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(sc) comparedto the 37° C. controls.

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 mM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

Solutions

Basolateral solution NaCl (135), CaCl₂ (1.2), (in mM): MgCl₂ (1.2),K₂HPO₄ (2.4), KHPO₄ (0.6), N-2- hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solution wastitrated to pH 7.4 with NaOH. Apical solution Same as basolateralsolution (in mM): with NaCl replaced with Na Gluconate (135).Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

2. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance>20 GΩ and a series resistance<15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 ml of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 mM forskolin and 20 mM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 □M of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 ΔM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(□F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Solutions

Intracellular solution Cs-aspartate (90), (in mM): CsCl (50) , MgCl₂ (1), HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted to 7.35 withCsOH). Extracellular solution N-methyl-D-glucamine (in mM): (NMDG)-Cl(150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35 with HCl).Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, □-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F⁻ (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing 2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Solutions

Extracellular solution NMDG (150), aspartic (in mM): acid (150) , CaCl₂(5) , MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).Intracellular solution NMDG-Cl (150), MgCl₂ (in mM): (2), EGTA (5), TES(10), and Tris base (14) (pH adjusted to 7.35 with HCl).Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

The compounds of the present invention were found to modulate CFTRactivity when tested using the above methods. The activity of exemplarycompounds of the present invention is recited below in Table 3.

EC50: “+++” means<10 uM; “++” means between 10 uM to 25 uM; “+” meansbetween 25 uM to 60 uM.

% Efficacy: “+” means<25%; “++” means between 25% to 100%; “+++”means>100%.

TABLE 3 Cmpd # EC50 uM % Activity 1 ++ ++ 2 +++ +++ 3 ++ ++ 4 +++ ++ 5++ ++ 6 ++ ++ 7 ++ +++ 8 +++ +++ 9 +++ ++ 10 ++ +++ 11 ++ +++ 12 +++ +++13 +++ ++ 14 +++ ++ 15 +++ ++ 16 +++ +++ 17 +++ ++ 18 ++ ++ 19 ++ ++ 20++ ++ 21 ++ ++ 22 ++ ++ 23 +++ ++ 24 ++ +++ 25 +++ ++ 26 ++ ++ 27 +++ ++28 ++ ++ 29 +++ ++ 30 +++ +++ 31 +++ ++ 32 ++ ++ 33 ++ ++ 34 ++ ++ 35 ++++ 36 ++ ++ 37 +++ +++ 38 +++ ++ 39 +++ +++ 40 +++ ++ 41 +++ ++ 42 +++++ 43 +++ ++ 44 ++ +++ 45 ++ ++ 46 +++ ++ 47 ++ ++ 48 +++ +++ 49 ++ ++50 +++ +++ 51 +++ +++ 52 +++ +++ 53 +++ +++ 54 ++ ++ 55 ++ ++ 56 +++ ++57 +++ ++ 58 ++ ++ 59 ++ ++ 60 ++ ++ 61 ++ ++ 62 +++ +++ 63 ++ +++ 64+++ +++ 65 ++ ++ 66 ++ ++ 67 ++ ++ 68 +++ ++ 69 ++ +++ 70 +++ +++ 71 +++++ 72 +++ +++ 73 ++ ++ 74 ++ ++ 75 ++ ++ 76 ++ +++ 77 ++ ++ 78 ++ ++ 79++ ++ 80 ++ ++ 81 ++ ++ 82 ++ ++ 83 ++ +++ 84 ++ ++ 85 ++ ++ 86 ++ ++ 87++ ++ 88 ++ +++ 89 +++ +++ 90 +++ +++ 91 +++ +++ 92 +++ ++ 93 +++ +++ 94+++ ++ 95 +++ ++ 96 ++ ++ 97 +++ +++ 98 +++ ++ 99 +++ +++ 100 ++ ++ 101+++ ++ 102 +++ +++ 103 +++ +++ 104 +++ ++ 105 +++ +++ 106 +++ ++ 107 ++++++ 108 +++ ++ 109 +++ ++ 110 +++ +++ 111 +++ +++ 112 +++ ++ 113 +++ ++114 +++ ++ 115 +++ +++ 116 +++ ++ 117 +++ +++ 118 +++ ++ 119 +++ +++ 120+++ ++ 121 +++ +++ 122 +++ +++ 123 +++ +++ 124 +++ ++ 125 +++ ++ 126 +++ 127 +++ ++ 128 +++ +++ 129 +++ ++ 130 +++ +++ 131 +++ +++ 132 +++ ++133 +++ ++ 134 +++ ++ 135 +++ ++ 136 +++ ++ 137 +++ ++ 138 +++ ++ 139+++ ++ 140 +++ ++ 141 ++ ++ 142 +++ ++ 143 + ++ 144 + ++ 145 ++ ++ 146+++ ++ 147 +++ ++ 148 +++ ++ 149 ++ ++ 150 ++ ++ 151 +++ ++ 152 ++ ++153 +++ ++ 154 +++ ++ 155 +++ ++ 156 +++ ++ 157 +++ ++ 158 +++4 ++ 159+++ ++ 160 ++ +++ 161 + ++ 162 + ++ 163 ++ ++ 164 ++ ++ 165 +++ +++ 166++ ++ 167 +++ ++ 168 +++ ++ 169 +++ ++ 170 +++ ++ 171 +++ ++ 172 +++ +++173 +++ ++ 174 +++ ++ 175 +++ ++ 176 +++ ++ 177 ++ ++ 178 +++ +++ 179 ++++ 180 ++ ++ 181 + ++ 182 + ++ 183 + ++ 184 ++ ++ 185 ++ ++ 186 ++ ++187 ++ ++ 188 + ++ 189 + ++ 190 + ++ 191 +++ ++ 192 ++ ++ 193 ++ ++194 + ++ 195 +++ ++ 196 +++ ++ 197 +++ ++ 198 +++ ++ 199 +++ +++ 200 +++ 201 +++ +++ 202 +++ ++ 203 +++ ++ 204 +++ ++ 205 +++ ++ 206 +++ ++207 +++ ++ 208 + ++ 209 +++ ++ 210 ++ ++ 211 +++ ++ 212 +++ ++ 213 ++ ++214 ++ ++ 215 +++ ++ 216 ++ ++ 217 + ++ 218 + ++ 219 + ++ 220 + ++ 221++ ++ 222 + ++ 223 + ++ 224 +++ ++ 225 +++ ++ 226 ++ ++ 227 ++ ++ 228 ++++ 229 ++ ++ 230 +++ +++ 231 ++ +++ 232 ++ ++ 233 + ++ 234 ++ ++ 235 +++++ 236 +++ ++ 237 +++ ++ 238 +++ +++ 239 +++ ++ 240 +++ ++ 241 +++ ++242 +++ +++ 243 +++ ++ 244 +++ ++ 245 +++ ++ 246 +++ ++ 247 +++ +++ 248+++ ++ 249 +++ ++ 250 +++ ++ 251 +++ ++ 252 ++ ++ 253 + ++ 254 +++ ++255 +++ ++ 256 +++ ++ 257 + ++ 258 ++ ++ 259 +++ ++ 260 +++ ++ 261 +++++ 262 + ++ 263 +++ ++ 264 + ++ 265 +++ +++ 266 +++ ++ 267 +++ ++ 268+++ ++ 269 +++ ++ 270 +++ ++ 271 +++ ++ 272 +++ +++ 273 +++ ++ 274 + ++275 + ++ 276 + ++ 277 + ++ 278 +++ ++ 279 + ++ 280 +++ ++ 281 ++ ++ 282+++ ++ 283 +++ ++ 284 +++ ++ 285 +++ ++ 286 +++ ++ 287 +++ ++ 288 +++ ++289 + ++

The invention claimed is:
 1. A method of modulating CFTR activity,comprising the step of contacting said CFTR with a compound of formula(II) or (III):

or a pharmaceutically acceptable salt thereof; wherein: X₁ is a bond, O,S, CF₂, CH₂, or NR; R is H or R² A₁ is aryl, or heteroaryl; each B₁ isindependently selected from 3-7 membered monocyclic, saturated,unsaturated or aromatic ring containing 0-4 heteroatoms selected from N,NH, S, or O; wherein each A₁ is optionally substituted with up to 4substituents independently selected from R¹, R², R³, R⁴, or R⁵; R¹ is R⁶or ((C1-C4)aliphatic)_(n)-Y; n is 0 or 1; Y is halo, CN, CF₃, OCF₃, OH,NH₂, NHR⁶, N(R⁶)₂, COOH, COOR⁶ or OR⁶; or two R¹ on adjacent ring atoms,taken together, form 1,2-methylenedioxy or 1,2-ethylenedioxy; R² isaliphatic, wherein each R² is optionally substituted with up to 2substituents independently selected from R¹, R⁴, or R⁵; R³ is acycloaliphatic, aryl, heterocyclic, or heteroaryl ring optionallysubstituted with up to 3 substituents, independently selected from R¹,R², R⁴ or R⁵; R⁴ is OR⁵, C(O)R⁵, C(O)OR⁵, C(O)R⁶, C(O)OR⁶, C(O)N(R⁶)₂,C(O)N(R⁵)₂, or C(O)N(R⁵R⁶); R⁵ is a cycloaliphatic, aryl, heterocyclic,or heteroaryl ring, optionally substituted with up to 3 R¹ substituents;and R⁶ is H or aliphatic provided that for compounds of formula (II):(i) when both B₁ are simultaneously phenyl and X₁ is CH₂, then A₁ is not4-fluoro-phenyl, 4-phenyl-piperidyl, phenyl, 2,4-dichloro-phenyl,4-methoxy-phenyl, 3,4-dichloro-phenyl, 2,5-dichloro-phenyl,4-nitro-phenyl, 4-bromo-phenyl, 4-methyl-phenyl, 2-chloro-phenyl,1-naphthyl, 3-trifluoromethyl-phenyl, 2,3-dichlorophenyl, N-morpholinyl,4-chloro-phenyl, 3-chloro-phenyl, or 3-nitro-phenyl; (ii) when X₁ is abond or CH₂, one B₁ is a substituted phenyl and the other B₁ iscycloaliphatic, then A₁ is not (C2-C8)aliphatic; and (iii) when X₁ is abond, then A₁ is not an optionally substituted 6-membered heteroarylring with 1-3 nitrogen ring atoms; and provided that for compounds offormula (III): (i) when X₁ is a bond, one B₁ is phenyl and the other B₁is N-piperidyl, then A₁ is not:

 and (ii) when X₁ is a bond, then A₁ is not an optionally substituted6-membered heteroaryl ring with 1-3 nitrogen ring atoms.
 2. The methodaccording to claim 1, wherein X₁ is CH₂, CF₂, or O.
 3. The methodaccording to claim 1, wherein X₁ is CH₂.
 4. The method according toclaim 1, wherein A₁ is selected from phenyl, triazinyl, pyrazinyl,pyrimidinyl, pyridazinyl, pyridyl, pyrazolyl, quinolinyl, isoquinolinyl,or naphthyl.
 5. The method according to claim 1, wherein each B₁ isindependently selected from optionally substituted C6-C10 aryl.
 6. Themethod according to claim 1, wherein each B₁ is independently anoptionally substituted phenyl or naphthyl.
 7. The method according toclaim 1, wherein each B₁ is an unsubstituted phenyl.
 8. The methodaccording to claim 1, wherein each B₁ is independently selected fromoptionally substituted C5-C12 heteroaryl.
 9. The method according toclaim 1, wherein each B₁ is independently selected from optionallysubstituted C5-C7 heteroaryl.
 10. The method according to claim 9,wherein each B₁ is independently selected from optionally substitutedpyrazolyl or imidazolyl.
 11. The method according to claim 1, wherein B₁is selected from optionally substituted pyrrolinyl, pyrazolinyl,piperidinyl, morpholinyl, or piperazinyl.
 12. The method according toclaim 1, wherein A₁ is optionally substituted C6-C10 aryl ring.
 13. Themethod according to claim 12, wherein A₁ is optionally substitutedphenyl or naphthyl.
 14. The method according to claim 1, wherein A₁ isoptionally substituted C5-C12 heteroaryl ring.
 15. The method accordingto claim 14, wherein A is pyridinyl.
 16. The method according to claim1, wherein each B₁ is independently an optionally substituted 3-12membered heterocyclic ring having up to 4 heteroatoms selected from O,S, or NR.
 17. The method according to claim 16, wherein each B₁ isindependently selected from optionally substituted pyrrolidyl,pyrrolinyl, pyranyl, piperidinyl, or morpholinyl.